`
`Special Article
`
`Monoclonal Antibody Therapy of Human Cancer: Taking
`the HER2 Protooncogene to the Clinic
`
`4 GAIL D. LEWIS, 1 JAY C. SARUP, 1 BRIAN M. FENDLY, 1 DANIEL
`H. MICHAEL SHEPARD, 1
`'
`MANEY AL, 1 JOYCE MORDENTI, 1 IRENE FIGARI, 1 CLAIRE E. KOTTS, 1
`MICHAEL A. PALLADINO, JR., 1 AXEL ULLRICH,2 and DENNIS SLAMON3
`
`Accepted: January 22, 1991
`
`The HER2 protooncogene encodes a 185-kDa transmem(cid:173)
`brane protein (pl85HER2) with extensive homology to the
`epidermal growth factor (EGF) receptor. Clinical and
`experimental evidence supports a role for overexpression
`of the HER2 protooncogene in the progression of human
`breast, ovarian, and non-sma11 cell lung carcinoma. These
`data also support the hypothesis that p1g5H£R2 present on
`the surface of overexpressing tumor cells may be a good
`target for receptor-targeted therapeutics. The anti(cid:173)
`pl85HER2 murine monoclonal antibody (muMAb) 405 is
`one of over JOO monoclonals that was derived following
`immunization of mice with cells overexpressing
`p185l:lER2 • The monoclonal antibody is directed at the
`extracellular (ligand binding) domain of this receptor
`tyrosine kinase and presumably has its effect as a result of
`modulating receptor function. In vitro assays have shown
`that muMAb 4D5 can specifically inhibit the growth of
`tumor cells only when they overexpress the HER2 pro(cid:173)
`tooncogene. MuMAb 4D5 has also been shown to en(cid:173)
`hance the TNF-cx sensitivity of breast tumor cells that
`overexpress this protooncogene. Relevant to its clinical
`application, muMAb 405 may enhance the sensitivity of
`pl85HER2-overexpressing tumor cells to cisplatin, a
`chemotherapeutic drug often used in the treatment of
`ovarian cancer. In vivo assays with a nude mouse model
`have shown that the monoclonal antibody can localize at
`
`1Department of Developmental Biology, Genentech, Inc., 460
`Point San Bruno Boulevard, South San Francisco, California
`94080.
`2Max Planck Institute for Biochemistry, Martinsreid, Germany.
`3Department of Hematology and Oncology, University of Cali(cid:173)
`fornia, Los Angeles, California 90024.
`"To whom correspondence should be addressed.
`
`the tumor site and can inhibit the growth of human tumor
`xenografts which overexpress p185HER2
`• Modulation of
`pl85HER2 activity by muMAb 405 can therefore reverse
`many of the properties associated with tumor progression
`mediated by this putative growth factor receptor. To(cid:173)
`gether with the demonstrated activity of muMAb 4D5 in
`nude mouse models, these results support the clinical
`application of muMAb 4D5 for therapy of human cancers
`characterized by the overexpression of pl85HER2.
`
`KEY WORDS: HER2; neu; TNF-o:; monoclonal antibody ther(cid:173)
`apy.
`
`BACKGROUND: THE HER2 PROTOONCOGENE
`AND HUMAN CANCER
`
`Ce!Jular protooocogenes encode proteins that are
`thought to regulate normal ceJJular proliferation and
`differentiation. Alterations in their structure or am(cid:173)
`plification of their expression lead to abnormal
`cellular growth and have been associated with car(cid:173)
`cinogenesis (1-4). Protooncogenes were first iden(cid:173)
`tified by either of two approaches. First, molecular
`characterization of the genomes of transforming
`retroviruses showed that the genes responsible for
`the transforming ability of the virus in many cases
`were altered versions of genes found in the genomes
`of normal cells. The normal version is the protoon(cid:173)
`cogene, which is altered by mutation to give rise to
`the oncogene. An example of such a gene pair is
`represented by the EGF receptor and the v-erbB
`gene product. The virally encoded v-erbB gene
`product has suffered truncation and other alter-
`
`117
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`SHEPARD ET AL.
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`ations that render it constitutively active and endow
`it with the ability to induce cellular transformation
`(5).
`The second method for detecting cellular trans(cid:173)
`forming genes that behave in a dominant fashion
`involves transfection of cellular DNA from tumor
`celJs of various species into nontransformed target
`cells of a heterologous species. Most often this was
`done by transfection of human, avian, or rat DNAs
`into the murine NIH 3T3 cell line (1-5). Following
`several cycles of genomic DNA isolation and re(cid:173)
`transfection, the human or other species DNA was
`molecularly cloned from the murine background
`and subsequently characterized. In some cases, the
`same genes were isolated following transfe.ction and
`cloning as those identified by the direct character(cid:173)
`ization of transforming viruses. In other cases,
`novel oncogenes were identifie.d. An example of a
`novel oncogene identified by this transfection assay
`is the neu oncogene. It was discovered by Weinberg
`and colJeagues in a transfection experiment in
`which the initial DNA was derived from a carcino(cid:173)
`gen-induced rat neuroblastoma (6,7). Characteriza(cid:173)
`tion of the neu oncogene revealed that it had the
`structure of a growth factor receptor tyrosine ki(cid:173)
`nase, had homology to the EGF receptor, and
`differed from its normal counterpart, the neu pro(cid:173)
`tooncogene, by an activating mutation in its trans(cid:173)
`membrane domain (8).
`The association of the HER2 protooncogene with
`cancer was established by yet a third approach, that
`is, its association with human breast cancer. The
`HER2 protooncogene was first discovered in cDNA
`libraries by virtue of its homology with the EGF
`receptor, with which it shares structural similarities
`throughout (5). When radioactive probes derived
`from the cDNA sequence encoding p185HER2 were
`used to screen DNA samples derived from breast
`cancer patients, amplification of the HER2 protoon(cid:173)
`cogene was observed in about 30% of patient sam(cid:173)
`ples (9). Further studies have confirmed this origi(cid:173)
`nal observation and extended it to suggest an
`important correlation between HER2 protoonco(cid:173)
`gene amplification and/or overexpression and wors(cid:173)
`ened prognosis in ovarian cancer and non-small cell
`lung cancer (10-14).
`The association of HER2 amplification/overex(cid:173)
`pression with aggressive malignancy, as described
`above, implies that it may have an important role in
`progression of human cancer; however, many tu(cid:173)
`mor-related cell surface antigens have been de(cid:173)
`scribed in the past, few of which appear to have a
`
`direct role in the genesis or progression of disease
`(15, l6). The data which support a role of HER2
`overexpression in the basic mechanisms of human
`cancer are summarized below.
`Amplified expression of pl85HER2 can lead to
`cellular transformation as assessed by morphologi(cid:173)
`cal alterations and growth of p185HER2-overex(cid:173)
`pressing celJs in soft agar and in nude mice (17, 18).
`In addition, NIH 3T3 fibroblasts overexpressing
`p185HER2 have an increased resistance to cytotox(cid:173)
`icity mediated by activated macrophages or recom(cid:173)
`binant human TNF-cx (19), the cytokine that ap(cid:173)
`pears to be mainly responsible for macrophage(cid:173)
`mediated tumor cell cytotoxicity (20). This
`observation extends also to breast tumor cells,
`which overexpress pl85HER2 (19), and suggests that
`high levels of pl85HER2 expression may be related
`to tumor cell resistance to at least one component of
`the host's antitumor surveillance armamentarium,
`the activated macrophage. This work has been
`reviewed previously (21), and similar data have
`recently been reported for ovarian tumor cell lines
`which overexpress p185HER2 (22). Further support
`for a role of p185HER2 or the related neu oncogene(cid:173)
`encoded tyrosine kinase in tumorigenesis comes
`from work with transgenic mice that have been
`manipulated to overexpress one or the other of
`these two related genes. Transgenic mice express(cid:173)
`ing the activated form of the rat neu protooncogeoe,
`under the control of a steroid inducible promoter,
`uniformly develop mammary carcinoma (23). In
`another transgenic mouse model the HER2 pro(cid:173)
`tooncogene product, ''activated'' by point mutation
`analogous to the rat neu oncogene product, or an
`unaltered form of the HER2 protooncogene, has
`been expressed in mice (24). The main malignancies
`induced in this model were either lung adenocarci(cid:173)
`noma or lymphoma but not mammary carcinoma.
`While it is not known why the different transgenic
`mouse models give such distinct results, the latter
`model may be of particular significance given the
`recent report of an association between p185HER2
`overexpression and poor prognosis in nonsmall celJ
`lung cancer (14). These differing results suggest
`some difference in the activity of activated neu and
`HERZ-encoded tyrosine kianses, although effects
`due to mouse strain differences cannot be excluded.
`The structural similarities between pl85HER2 and
`the EGF receptor suggest that function of p185HER2
`may be regulated similarly to the EGF receptor. In
`particular, one expects that the tyrosine kinase
`activity associated with the cytoplasmic domain of
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`Journal of Clinical Immunology, Vol. 1I, No. 3, 1991
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`Fig. 1. Suppression of the TNF cytotoxic response by activation of p 1 g5HER2. The schematic shows both the TNF
`cytotoxic pathway (top) and the pl85HER2-stimulated cell proliferation/transformation pathway (bottom). Signaling
`from the TNF receptor following interaction with TNF has not been characterized. Binding of ligand to p185HER2
`is shown to activate the receptor-associated tyrosine kinase activity, resulting in stimulation of cellular
`proliferation and suppression of the tumor cell cytotoxic response to TNF.
`
`the receptor would be ligand activated. This pro(cid:173)
`posal receives support from recent work describing
`a ligand for p185HER2 (25). These data lead to a
`model (Fig. 1) wherein antagonists that downregu(cid:173)
`late the function of p185HER2 should have the effect
`of inhibiting growth of tumor ce!Js dependent upon
`pJ85HER2 function and of increasing the sensitivity
`of such tumor cells to TNF-o:. By analogy with
`previous work done with two related tyrosine ki(cid:173)
`nases, the EGF receptor (26) and the activated neu
`protooncogene product (27), we hypothesized that
`monoclonal antibodies targeted to the extracellular
`domain of p185HER2 may have the desired proper(cid:173)
`ties.
`
`DERIVATION OF muMAb 4D5
`
`A family of monoclonal antibodies focused
`against the extracellular domain of pl85HER2 were
`prepared (28). To do this, an NIH 3T3 fibroblast cell
`line that overexpresses pl85HER2 [NIH 3T3/HER2-
`3-400 (18)] was used to immunize BALB/c mice.
`The mice were subsequently boosted with NIH
`3T3/HER2-3-400 and, finally, with a preparation
`
`enriched for pl85HER2 by wheat germ agglutinin
`chromatography of membrane extracts of this cell
`line. Following splenocyte fusion with a mouse
`myeloma partner, the hybridomas were cultured in
`96-well microtiter plates. Hybridomas positive for
`anti-p 1s5HER2 activity, but with little or no anti(cid:173)
`EGFR activity, were detected by ELISA (Fig. 2). A
`critical property of an anti-p185HER2 monoclonal
`antibody with potential for therapy would be its
`lack of cross-reactivjty with the closely related
`EGF receptor, which is expressed at elevated levels
`in multiple tissues (29). To select further monoclo(cid:173)
`nal antibodies with this characteristic, a number of
`assays were performed, including immunoprecipita(cid:173)
`tion assays utilizing in vivo labeled EGF receptor
`and p1s5HER2 (Fig. 3A) and FACS analysis of
`antibody binding to tumor cells overexpressing ei(cid:173)
`ther p185HER2 or the EGFR (Fig. 3B). The screen(cid:173)
`ing results are summarized in Table I. Based upon
`these results, nine of the p185HER2 monoclonal
`antibodies were chosen for further characte1ization,
`including a cell growth inhibition assay utilizing the
`SK-BR3 human breast adenocarcinoma cell line,
`which greatly overexpress p185HER2 . The monoclo-
`
`.Journal of Clinical Immunology, Vol. II, No. 3, 1991
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`3 of 11
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`120
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`SHEPARD ET AL.
`
`1.B
`
`1.6
`
`1.4
`
`1.2
`
`1.0
`
`O.B
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`E
`c
`N
`0)
`
`""'
`0
`0
`
`2C4
`
`7C2
`
`7F3
`
`2H11
`
`3H4
`
`3EB
`
`405
`
`SBB
`
`6E9
`
`703
`
`586 40 .1H1
`
`MAb (1 ug/ml)
`Fig. 2. ELISA screening of anti-pl85HERi monoclonal antibodies. Results shown measure the relative
`reactivities of the purified anti-pl85HER2 monoclonal antibodies (added to l µ,g/ml) with membrane extracts
`enriched in EGF receptor (open bars; from A43l squamous carcinoma cells) or enriched in pl85ffER2 (filled
`bars; from NlH 3T3/HER2-3-400).
`
`nal antibody, muMAb 405, was clearly the most
`effective of the group in this assay (Table II).
`The initial results characterizing the growth in(cid:173)
`hibitory activity of these monoclonal antibodies
`were extended by comparing them for activity
`against a battery of human breast and ovarian tumor
`cell lines that expressed varying levels of p 185HER2.
`These results reveal that the monoclonal antibodies
`can be growth inhibitory, they may have no affect
`on cell proliferation, or they may stimulate the
`proliferation of breast tumor cells. Gwwth inhibi(cid:173)
`tion appears to depend upon overexpression (Table
`III). This property, in particular, is shared by the
`monoclonal antibodies 405 and 3H4. These mono(cid:173)
`clonal antibodies may exert their effects on cell
`growth by similar mechanisms since they compete
`for binding to the receptor (Tables I and Ill) (28)
`and, therefore, may recognize the same or overlap(cid:173)
`ping epitopes. The other monoclonal antibodies
`vary in their ability to inhibit proliferation, but 7C2
`and 6E9 are consistently less active in this respect.
`
`The potent growth inhibitory activity of 2C4 for
`MDA-MB-175 breast tumor cells is not understood
`at present but may represent cross-reactivity with
`another receptor expressed on these cells. Simi(cid:173)
`larly, the properties that distinguish 7C2 from the
`other antibodies with regard to its ability to stimu(cid:173)
`late the proliferation of several of the tumor cell
`lines shown in Table III has not been determined.
`The 6£9 monoclonal antibody has been shown to
`bind to the extracellular domain of pl85HER2 , al(cid:173)
`though only to a subset of receptors on the surface
`of SK-BR-3 tumor cells (30). The functional signif(cid:173)
`icance of this subset of receptors is unclear. In
`addition to its activity on breast tumor cells, which
`overexpress p185HER2, muMAb 405 is also clearly
`the most active of the monoclonal antibodies with
`respect to its ability to inhibit growth of SKOV-3, a
`human ovarian adenocarcinoma cell line that over(cid:173)
`expresses p185HERi (Table Ill). Experiments are
`currently planned to try to understand in more
`detail how these monoclonal antibodies may exert
`
`Journal of Clinical Immunology, Vol. 11, No. 3, 1991
`
`4 of 11
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`Celltrion, Inc., Exhibit 1048
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`MONOCLONAL ANTIBODY THERAPY OF HUMAN CANCER
`
`121
`
`I
`
`\
`
`~ i t /\ !
`\ ;
`! '
`
`'
`
`A.
`
`8.
`
`CVN
`
`HER1
`
`.,....
`~
`ci
`v
`4
`
`I/)
`0
`v
`5
`
`co
`0
`
`6
`
`I/)
`0
`v
`2
`
`co
`0
`
`3
`
`.,....
`::i::
`0
`ci
`v
`1
`
`HER2
`.,....
`::i::
`ci
`v
`7
`
`co
`0
`
`9
`
`I/)
`0
`v
`8
`
`185
`-
`-110
`
`-
`
`0 ....
`Q)
`.0
`E
`:::J z
`~
`~
`Q)
`CI:
`
`!
`
`I
`
`104
`103
`102
`10
`0.1
`Relative Fluorescence Intensity
`Fig. 3. MuMAb 4D5 does not cross-react with the EGFR. (A) Iromunoprecipitation of metabolically Jabelelled NIH 3T3 cells
`transfected by control plasmid (CVN), by a plasmid encoding the EGFR (HERi) or a plasmid encoding pl85HER2 (HER2).
`MuMAb 40. l.HI is directed against hepatitis B surface antigen (lanes I, 4, 7); muMAb 4D5 is directed against pl85H£Rz
`(lanes 2, 5, 8); muMAb 108 is directed against the EGFR (lanes 3, 6, 9). (B) Fluorescence-activated cell sorter histograms
`of muMAb 40.1.Hl (solid lines) or muMAb 4D5 (dotted lines) reacted with SK-BR-3 tumor cells (approx. 2 x 106 receptors
`per cell; upper panel) or the same antibodies reacted with the A431 squamous carcinoma cell line (approx. 2 x 106 EGFR
`per cell; lower panel).
`
`distinct effects on tumor cell proliferation. The in
`vitro results summarized in Table Ill clearly show
`that when the monoclonal antibodies are compared
`for efficacy, as measured by their abilities to inhibit
`growth of breast and ovarian tumor cells overex(cid:173)
`pressing p185HER2, muMAb 4D5 is usually the most
`potent and is therefore a good candidate for further
`characterization in other models that may be pre(cid:173)
`dictive of its efficacy in human clinical trials. Inter(cid:173)
`estingly, the most dramatic activity of the antibody
`is seen in cell lines that overexpress greater than
`fivefold the level observed in MCF-7 breast tumor
`cell lines [a low expressor control cell line; Table III
`(19)]. Patients who overexpress greater than five(cid:173)
`fold the normal level of p 1 g5HER2 have been shown
`to have a very poor prognosis (10). These results
`will aid in choosing patients who are most likely to
`respond in clinical trials.
`The model depicted in Fig. 1 predicts that down(cid:173)
`regulation of pl85HER2 by a monoclonal antibody or
`
`other reagent should result in decreased ceUular
`proliferation, as shown in Table III, but also in(cid:173)
`creased sensitivity to TNF-a. Results of experi(cid:173)
`ments in which tumor cells overexpressing
`pl85HER2 were treated with muMAb 4D5 or a
`control monoclonal antibody, alone and in combi(cid:173)
`nation with TNF-a, suggest the validity of this
`model (Fig. 4) (31). MuMAb 4D5 treatment of
`breast tumor cells overexpressing p 1 g5HER2 re(cid:173)
`sulted in enhanced sensitivity of these cells to
`TNF-a. The growth and the TNF-a sensitivity of
`normal cells or tumor cells that do not overexpress
`the receptor were unaltered.
`In addition to the relationship between TNF-a
`resistance and p185HER2 overexpression, a possible
`relationship between protoncogene expression and
`resistance to the chemotherapeutic drug cisplatin
`has been investigated. A correlation between HER2
`protooncogene overexpression and resistance to
`chemotherapeutic drugs rests on the grounds that
`
`Journal of Clinical Immunology, Vol. 11, No. 3, 1991
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`5 of 11
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`Celltrion, Inc., Exhibit 1048
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`Table 1. Summary Table of Monoclonal Antibodies Described
`RIP"
`
`SHEPARD ET AL.
`
`122
`
`MAb
`
`4D5
`2C4
`2Hll
`3E8
`3H4
`SB8
`6E9
`7C2
`703
`7F3
`
`EGFR
`
`Isotype
`
`IgGl ,k
`IgGl ,k
`IgG2a,k
`Ig02a,k
`IgGl,k
`IgGl ,k
`lgGl,k
`IgGJ,k
`IgGl,k
`JgGl ,k
`
`ELISA0
`pJ85HER2
`
`+ +
`+ ++
`+
`+
`+
`+
`+ +
`++
`+ +
`+ ++
`
`EGFR
`
`pl85HER2
`
`Epitopec
`
`FACSd
`
`++
`+ +
`++
`++ +
`+
`+ +
`+
`++
`+ +
`+ ++
`
`I(p/c)
`F(p/c)
`H(p/c)
`H(p/c)
`l(p)
`nd(p)
`nd(p)
`G(p)
`F(p/c)
`G/F(p/c)
`
`++ +
`+ ++
`+ +
`+ ++
`+
`+
`
`+ + +
`+++
`+ ++
`
`•summary of OD 492 nm: (-) < 0.1 ; (+) 0.11-0.50; (+ +) 0.51-1.0; ( + ++) > 1.0.
`bSummary of autoradiography from immunoprecipitations: (-) bands equal to negative control; ( +) weak bands but darker than negative
`control; ( + + ) moderately exposed bands; ( + + +) strongly exposed bands.
`ci..etters were assigned to represent individual epitopes A through I (nd, not done). MAbs were considered to share an epitope if each
`blocked binding of the other by 50% or greater in comparison to an irrelevant MAb control. The epitope composition recognized by
`immunoprecipitations with each MAb from tunicamycin-treated cells is shown. The letters p, c, or p/c in parentheses indicate that the
`monoclonal antibody binds only to the polypeptide (p), the carbohydrate (c), or both (p/c) moieties in the extracellular domain of
`p!85HER2.
`dfluorescence staining of SK-BR-3 cells by the anti-pl85HER2 monoclonal antibodies:(-) MAbs equal to the negative control MAbs; ( +)
`1- to 9-fold higher than the negative controls;(++) 10· to 99-fold higher than the negative controls; (+++) > 100-fold higher than the
`negative controls.
`
`patients exhibiting overexpression appear to have a
`worsened prognosis, especially in ovarian cancer
`(10, 13). In addition, recent work with the EGF
`receptor (32) has indicated that when the anti(cid:173)
`EGFR monoclonal antibody 108.4 was added to(cid:173)
`gether with cisplatin, the antitumor effect of the
`antibody was greatly enhanced. Because the 108.4
`monocJonal antibody and muMAb 4D5 appear to
`share the ability to inhibit soft agar growth of tumor
`
`Table II. Inhibition of SK-BR-3 Proliferation by Anti-pl85HBRZ
`Monoclonal Anlibodies0
`
`Monoclonal
`antibody
`
`Relative cell
`proliferationb
`
`44.2 ± 4.4
`79.3 ± 2.2
`79.5 ± 4.4
`83.8 ± 5.9
`66.2 ± 2.4
`98.9 ± 3.6
`62.l ± 1.4
`66.5 ± 3.9
`92.9 ± 4.8
`105.8 ± 3.8
`94.7 ± 2.8
`
`4D5
`7C2
`2C4
`7D3
`3E8
`6E9
`7F3
`3H4
`2Hll
`40.1 Hie
`4F4
`0 SK-BR-3 breast tumor cells were plated at a density of 4 x Hf
`cells per well into 96-well microdilution plates, allowed to
`adhere, and then treated with monoclonal antibody (10 µg/ml).
`bRelative cell proliferation was determined by crystal violet
`staining of the monolayers after 72hr. Values are expressed as a
`percentage of results with untreated control cultures (100%).
`ccontrol monoclona.1 antibodies 40. lHI and 4F4 are directed
`against hepatitis B surface antigen and human interferon-"(,
`respectively (27).
`
`cells overexpressing their respective receptors, it
`seemed possible that such an interaction may also
`occur in the HER2 protooncogene system. The in
`vitro results (Fig. 5) show that treatment of SK(cid:173)
`BR-3 breast tumor cells with muMAb 4D5 enhances
`their sensitivity to cisplatin.
`
`IN VIVO PRECLINICAL EFFICACY
`
`A critical part of the rationale supporting the
`application of muMAb 4D5 to human cancer ther(cid:173)
`apy is its ability to inhibit the growth of tumor cells
`overexpressing p185HER2 in vivo. A human tumor
`xenograft model was used to test this property of
`muMAb 405 and to compare its activities with
`those of the other monoclonal antibodies in a rele(cid:173)
`vant model of human disease. In this model, a
`human breast tumor, characterized with respect to
`HER2 protooncogene amplification and expression,
`was grafted into the subrenal capsules of nude mice.
`Therapy was initiated t week postimplantation. In
`order to be active in this model, the monoclonal
`antibody must be able to localize to the overex(cid:173)
`pressing tumor cells in the lesion and subsequently
`exert a growth regulatory effect mediated through
`pl85HER2. Growth inhibition occurs only with tu(cid:173)
`mors that overexpress the receptor. Heterotrans(cid:173)
`plants (approximately 1 mg) of Murray breast tumor
`[a high expresser of the HER2 gene product (10)]
`were implanted into the subrenal capsule of 48
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`Journal of Clinical Immunology, Vol. I 1, No. 3, 1991
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`MONOCLONAL ANTIBODY THERAPY OF HUMAN CANCER
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`123
`
`Table Ill. Inhibition of Human Breast and Ovarian Tumor Cell Growth by Monoclonal Antibodies Directed Against the Extracellular
`Domain of pJg5HER2
`
`Cell line
`
`MCF7
`ZR-75-1
`MDA-MB-175
`MDA-MB-453
`MDA-MB-361
`BT474
`SK-BR-3
`SK-OV-3
`
`Relative
`pJ85HER2
`expression°
`
`l
`3
`4
`7
`17
`20
`33
`17
`
`Cell proliferation (% control)b
`
`4D5c
`
`3H4c
`
`2C4d
`
`7F3d
`
`7C2'
`
`6E9'°
`
`94
`106
`61
`62
`60
`23
`42
`77
`
`101
`ll3
`84
`68
`68
`25
`56
`85
`
`101
`104
`24
`91
`65
`53
`66
`87
`
`97
`100
`48
`84
`73
`20
`64
`91
`
`106
`149
`87
`78
`ll3
`74
`92
`97
`
`110
`113
`103
`101
`J13
`94
`105
`99
`
`"Based on FACS assay using muMAb 405 and fluorescence-labeled goat anti-murine lgGJ polyclonal antibody.
`bFive-day assay with 10 µg/ml of indicated monoclonal antibody (SE, - 10%). Other methods as described in the footnotes to Table II.
`c405 and 3H4 define epitope " [. "
`d2C4 and 7F3 will partially block one another, 2C4 is assigned epitope "F," and 7F3 is assigned epitope "F/G. "
`'7C2 defines epitope " G" and will partially block 7F3 binding.
`f6E9 epitope determination not done.
`
`athymic mice on day 0. Groups of eight animals
`were injected intravenously with tissue culture(cid:173)
`derived muMAb 4D5 (36.4 mg/kg), PBS, or control
`monoclonal antibody , muMAb 5B6 (directed
`against gpl20; 36.4 mg/kg), as single agents in
`equally divided doses on days 7, 10, and 13. Four
`mice from each group were sacrificed on day 20,
`and the remainder of the animals were sacrificed on
`day 34. Tumor sizes were measured using ocular
`micrometer and gravimetric techniques. A sum(cid:173)
`mary of the tumor weights (mean ± SD) from
`animals sacrificed on days 20 and 34 is shown in
`Table IV. On day 20, average tumor weights of
`animals receiving muMAb 4D5 were significantly
`less than those receiving the same dose of the
`control antibody muMAb 5B6. Interactive effects
`between muMAb 4D5 and cisplatin have also been
`observed in this model (33). These studies in athy(cid:173)
`mic mice bearing human breast tumor xenografts
`have demonstrated efficacy and suggested an en(cid:173)
`hanced effect when muMAb 4D5 is given in combi(cid:173)
`nation with cisplatin.
`
`a modulation of intracellular second messengers,
`including diacylglycerol. Diacylglycerol (DAG) is a
`product of phospholipase C breakdown of phos(cid:173)
`phatidylinositol-4,5-bisphosphate. It is a cofactor
`
`A SK·BR-3
`
`B SK-BR-3
`
`C. MDA-MB-175-Vll
`
`1.5
`
`1.0
`
`D. MDA·MB-231
`
`E. HBL-100
`
`F. T24
`
`0.5
`
`c
`0
`·~ cu
`....
`~
`0 ....
`a..
`Q)
`{.)
`Cl> >
`·~ 1.0
`Q)
`a:
`
`0.0
`1.5
`
`0.5
`
`0.0
`
`MECHANISM OF ACTION
`
`The results described above are consistent with
`muMAb 4D5 having receptor antagonist activity.
`Surprisingly, however, muMAb 4D5 treatment of
`SK-BR3 tumor cells stimulates receptor tyrosine
`kinase activity (Table V) (30, 34). In addition, it can
`mediate the phosphorylation of intracellular sub(cid:173)
`strates by pl g5HER2 (34). Consistent with its ability
`to stimulate receptor activity, muMAb 4D5 treat(cid:173)
`ment of SK-BR-3 or SK-OV-3 tumor cells results in
`
`Fig. 4. Monoclonal antibody 405 sensitizes breast tumor cells to
`the cytotoxic effects of TNF-a. Filled bars, cell number at
`initiation of the assay; dark cross-hatching, untreated control;
`dark stipples, TNF-a alone; light cross-hatching, MuMAb 405;
`open bars, MuMAb 405 combined with TNF-a. (B) Lack of
`growth inhibition of SK-BR3 tumor cells by nmMAb 40.l HI
`(anti-hepatitis B antigen; light stipples) and failure of the 40. l H
`l to enhance SK-BR-3 tumor cell sensitivity to TNF-a (broken
`cross-hatching). SK-BR-3 and MDA-MB-175-VII overexpress
`p185HER2 (see Table lll). MDA-MB-231 and HBL-100 are
`breast cell lines which do not overexpress pl85HER2, and 1'24 is
`a nonoverexpressing human bladder carcinoma cell line. The
`assay was performed as described in Ref. 31.
`
`Journal of Clinical Immunology, Vol. 11, No. 3, 1991
`
`7 of 11
`
`Celltrion, Inc., Exhibit 1048
`
`
`
`124
`
`SHEPARD ET AL.
`
`8
`
`~
`
`140 A
`~ 120
`:0 co 100 Ol
`-
`5
`c
`Q)
`~
`Q)
`0..
`Q)
`>
`".;:! ro
`Q)
`a:
`
`~
`
`6;
`
`80
`
`60
`
`40 ~
`
`20
`
`0
`
`0
`
`.01
`
`1
`.1
`Cisplatin (µg/ml)
`
`10
`
`100 0
`
`.01
`
`10
`1
`.1
`Cisplatin (µg/ml)
`
`''"I
`100
`
`Fig. 5. Treatment of SK-BR-3 breast tumor cells with muMAb 405 enhances sensitivity to
`cisplatin. MuMAb 405 (A) or muMAb 6E9 (control; B) and cisplatin were added at the indicated
`concentrations to SK-BR-3 breast tumor cells. The plate cultures were incubated for 3 days and
`relative cell proliferation was determined as described (31). No antibody (0); 0.156 µ.g/ml muMAb
`405 or muMAb 6E5' (• ); 0.625 µ.g/ml muMAb 405 or muMAb 6E9 (t:.); 2.5 µ.g/ml muMAb 4D5
`or muMAb 6E9 (A).
`
`for activation of protein kinase C and has been
`closely associated with growth factor activity (:55).
`As may be predicted from its effect on cell prolifer(cid:173)
`ation, muMAb 4D5· treatment of SK-BR-3 tumor
`cells results in downregulation of intracellular pools
`of DAG (Table VO (30). This result is consistent
`with overall antagonist activity, as is inhibition of
`tumor cell proliferation. Other data suggest that
`muMAb 405 may inhibit association of ligand with
`the receptor (25). Similar monoclonal antibodies
`have been reported for the EGFR system (26, 28).
`Further work is under way to characterize the
`ligand(s) that binds p185HER2 and the mechanism of
`action of muMAb 4D5.
`While the ability of muMAb 405 to stimulate
`phosphorylation of pl 85HER2 is consistent with an
`agonist of receptor function, it is important to note
`that our current data suggest that it does not behave
`as an agonist in our cell growth assays in vitro or in
`nude mice. The results of an experiment that com(cid:173)
`pares the effects of muMAb 4D5 on the growth of
`
`MCF-7 and SK-BR-3 breast tumor cells are shown
`in Fig. 6. These data demonstrate that muMAb 4D5
`has no effect on the growth of nonoverexpressing
`tumor cells (MCF-7; Tables Ill, VII) at any of the
`doses tested between 0.7 pM and 67 nM. Also,
`whatever allows the muMAb 4D5 to have differen(cid:173)
`tial effects on overexpressing tumor cells, this dif(cid:173)
`ference does not lie in different receptor affinities
`for the monoclonal antibody. Table VII clearly
`shows that SK-BR-3 and SK-OV-3, both p185HER2
`overexpressors, which are growth inhibited by 405,
`and MCF-7, which is not, all have similar affinities
`for muMAb 4D5. The clearest difference between
`these cell lines is the number of binding sites per
`tumor cell. These data are consistent with other
`work that has been previously reported with tumor
`cells in monolayer culture or in soft agar (18, 19,
`25).
`A possible mechanistic explanation, which takes
`many of our experimental observations into ac(cid:173)
`count, is a model in which muMAb 4D5 binds
`
`Table IV. MuMAb 405 [nhlbits the Growth of a Human Breast
`Tumor (Murray) in Athymic Mice0
`
`Table V. Effect of muMAb 405 on Phosphoamino Acid
`Content of p185t1ER2 in SK-BR-3 Cells
`
`Tumor weight (mg)"
`
`Group (n = 4)
`
`Day 20
`
`Day 34
`
`PBS
`Control .(gG (muMAb5B6)
`muMAb4D5
`
`6.79 :!: 9.79
`7.11 :!::5.48
`1.48 ::!:: 1.10
`
`36.0 ± 30.7
`88. l ± 91.4
`6.5 :!: 6.4
`
`Phospho-
`tyrosine
`cpm0 %b
`
`Phospbo-
`serine
`%
`
`cpm
`
`Treatment
`
`None
`muMAb4D5
`
`11
`827
`
`1.5
`14.0
`
`564
`3,658
`
`75
`62.0
`
`Phospho-
`threonine
`
`cpm
`
`176
`1,429
`
`%
`
`23.5
`24.0
`
`0 Administered as equally-divided intravenous doses on days 7,
`IO, and 13 post tumor implantation.
`hData are mean :t standard deviation (SD) (n = 4).
`
`0 Phosphoamino acids as cpm are expressed following back(cid:173)
`ground subtraction (17 cpm for none, 21 cpm for muMAb 405).
`6Percentage of total phosphoamino acids.
`
`J.ournal of Clinical Immunology, Vol. 11, No. 3, 1991
`
`8 of 11
`
`Celltrion, Inc., Exhibit 1048
`
`
`
`MONOCLONAL ANTIBODY THERAPY OF HUMAN CANCER
`
`125
`
`Table VJ. Effect of muMAb 4D5 or 6E9 Monoclonal
`Antibodies on sn-1 ,2-diacylglycerol levels in SK-BR-3 Cells
`
`Table Vll. Monoclonal Antibody Binding to Cultured Human
`Adenocarcinoma Cells
`
`Time
`
`5 min
`5 min
`5 min
`
`24 hr
`24 hr
`24 hr
`
`Treatment
`
`Vehicle
`muMAb4D5
`muMAb6E9
`
`Vehicle
`muMAb4D5
`muMAb6E9
`
`pmol sn-1,2-
`DAG/106 cells
`
`111.0 ± 10
`133.2 ± 11.3
`133.3 ± 12.9
`
`98.6 ± 9.6
`62.1 ± 7.4
`92.0 ± 12.7
`
`0
`+ 20
`+ 20
`
`0
`- 37
`- 7
`
`% change
`
`Cell line
`
`Antibody Kd(nM)
`
`SK-BR-3 muMAb4D5
`SK-OV-3 muMAb4D5
`MCF7
`muMAb4D5
`
`6.0
`5.0
`1.2
`
`Receptor no. Growth
`inhibitionn
`(sites/cell)
`- *
`-*
`
`926,650
`428,930
`5,525
`
`nFollowing incubation with monoclonal antibody (33 nM) or
`vehicle (PBS) control, the reactions were terminated by aspi(cid:173)
`rating the media and adding 1 ml of ice-cold 100% MeOH. Cells
`were scraped from the plates and transferred to 13 x 100-mm
`glass tubes containing 1 ml I 00% chloroform. Plates were rinsed
`with an additional 1 ml of cold methanol, and the rinses
`combined and mixed thoroughly. Following phase separation at
`room temperature for 30 min, 1 ml methanol and I ml NaCl were
`added, the samples were centrifuged at 3000 rpm for 5 min, and
`the top aqueous layer was aspirated. The remaining organic
`phase was assayed for sn-1,2-diacylglycerol by standard proce(cid:173)
`dures.
`
`tightly to p I g5HER2, excludes ligand binding, stim(cid:173)
`ulates receptor internalization, and downregulates
`receptor signaling pathways as a result of constitu(cid:173)
`tive activation of tyrosine kinase activity that re(cid:173)
`sults from nondissociation of the muMAB 4D5/
`p 185HER2 complex during receptor cycling. This
`hypothesis has additional support from our obser-
`110 i
`100 fFI
`90 i
`
`I
`
`80 '
`
`70
`
`60
`
`50
`
`40 ~
`30 J
`'
`20 1/
`0 .0001 .001
`
`.01
`
`.1
`
`10 100
`
`c
`0
`"+::
`('Cl
`.....
`~
`0 ....
`a.
`Qi
`()
`<l>
`
`> "fJ
`a;
`a:
`
`405 (nM)
`Fig. 6. MuMAb 4D5 does not stimula