United States Patent [19]
`Dudziak et al.
`
`1111
`
`~111111~11~ 1111111111111111111111111111111
`US005677171A
`5,677,171
`[11] Patent Number:
`[45] Date of Patent:
`Oct. 14, 1997
`
`[54] MONOCLONAL ANTffiODIES DIRECTED
`TO THE HER2 RECEPTOR
`
`[75]
`
`Inventors: Robert M. Hudziak, Corvallis, Oreg.;
`H. Michael Shepard, Rancho Santa Fe,
`Calif.; Axel Ullrich, Portola Valley,
`Calif.; Brian M. Fendly, Half Moon
`Bay, Calif.
`
`[73] Assignee: Genentech, Inc., South San Francisco,
`Calif.
`.
`
`[21] Appl. No.: 286,303
`
`[22] Filed:
`
`Aug. 5,1994
`
`Related U.S. Application Data
`
`[63] Continuation of Ser. No. 977,453, Nov. 18, 1992, aban(cid:173)
`doned, which is a continuation of Ser. No. 147,461, Jan. 25,
`1988, abandoned, which is a continuation-in-part of Ser. No.
`143,912, Jan. 12, 1988, abandoned.
`Int. CI.6 ••••••••••••••••.•••••••• C07K 16/00; GOlN 33/574
`[51]
`[52] U.S. Cl ................................... 435/240.27; 530/388.8;
`530/388.85; 530/387.7; 435n.23; 435/172.2
`[58] Field of Search .............................. 530/387.7, 388.8,
`530/388.85, 381.1; 435n.23, 240.27, 112.2,
`70.21
`
`[56]
`
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`(1985).
`
`(List continued on next page.)
`
`Primary Examiner-Lila Feisee
`Assistant Examiner-Geetha P. Bansal
`Attorney, Agent, or Firm-Wendy M. Lee
`
`[57]
`
`ABSTRACT
`
`Monoclonal antibodies which bind to the extracellular
`domain of the HER2 receptor and inhibit growth of
`SK-BR-3 breast tumor cells, which overexpress HER2, are
`disclosed. The monoclonal antibodies can be used for in
`vitro assays for detecting a tumor characterized by amplified
`expression of HER2.
`
`39 Claims, 6 Drawing Sheets
`
`PFIZER EX. 1096
`Page 1
`
`

`

`5,677,171
`Page 2
`
`OTHER PUBLICATIONS
`
`Masui et al., "Mechanism of Antitumor Activity in Mice for
`Anti-Epidermal Growth Factor Receptor Monoclonal Anti(cid:173)
`Isotypes" Cancer Research
`bodies with Different
`46;5592-5598 (1986).
`Morrison, ''Transfectomas Provide Novel Chimeric Anti(cid:173)
`bodies" Science 229:1202-1207 (1985).
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`Induced by the Transforming DNA of Rat Neuroblastomas"
`Cell28:865-871 (1982).
`Rodeck et al., ''Interactions between growth factor receptors
`and corresponding monoclonal antibodies in human tumors"
`J. Cellular Biochem. 35(4):315-320 (1987).
`Rodeck et al., ''Tumor Growth Modulation by a Monoclonal
`Antibody to the Epidermal Growth Factor Receptor: Immu(cid:173)
`nologically Mediated and Effector Cell-independent
`Effects" Cancer Research 47(14):3692-3696 (1987).
`Rosenthal et al., "Expression in Rat Fibroblasts of a Human
`Transforming Growth Factor-a eDNA Results in Transfor(cid:173)
`mation" Cell46:301-309 (1986).
`Sato et al., "Biological Effects in Vitro of Monoclonal
`Antibodies to Human Epidermal Growth Factor Receptors"
`Mol. Bioi. Med. 1:511-529 (1983).
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`Gene Distinct from and Unliked to the Gene Encoding the
`EGF Receptor" Science 229:976-978 (1985).
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`clonal Antibodies on the Management of Carcinomas: The
`Richard and Hinda Rosenthal Foundation Award Lecture"
`Cancer Research 46:3225-3238 (1986).
`Sem.ba et al., "A v-erbB-related protooncogene c--erbB-2,
`is distinct from the c-erb-B-1/epiderrnal growth factor(cid:173)
`receptor gene and is amplified in a human salivary gland
`adenocarcinoma" Proc. Natl. Acad. Sci. USA 82:6497-6501
`(1985).
`Slamon et al., "Human Breast Cancer: Correlation of
`Relapse and Survival with Amplification of the HER-2/neu
`Oncogene" Science 235:177-182 (1987).
`Sobol et al., "Epidermal Growth Factor Receptor Expression
`in Human Lung Carcinoma Defined by a Monclonal Anti(cid:173)
`body'' J. Natl. Cancer Institute 79(3):403-407 (1987).
`Sugarman et al., "Effects of Growth Factors on the Antipro(cid:173)
`liferative Activity of Tumor Necrosis Factor" Cancer
`Research 47:780-786 (1987).
`Sugarman et al., "Recombinant Human Tumor Necrosis
`Factor-a: Effects on Proliferation of Normal and Trans(cid:173)
`formed Cells in Vitro" Science 230:943-945 (1985).
`Takahashi et al., "Radioirnmunodetection of Human Glioma
`Xenografts by Monoclonal Antibody to Epidermal Growth
`Factor Receptor" Cancer Reserch 47:3847-3850 (1987).
`Urban et al., ''Tumor necrosis factor: A potent effector
`molecule for tumor cell killing by activated macrophages"
`Proc. Natl. Acad. Sci. 83:5233-5237 (1986).
`Van de Vijver et al., "Amplification of the neu (c--erbB-2)
`Oncogene in Human Mammary Tumors Is Relatively Fre(cid:173)
`quent and Is Often Accompanied by Amplification of the
`Linked c--erbA Oncogene" Molecular & Cellular Biology
`7(5):2019-2023 (1987).
`Venter et al., "Overexpression of the c--erbB-2 Oncoprotein
`in Human Breast Carcinomas: Immunohistological Assess(cid:173)
`ment Correlates with Gene Amplification" Lancet pp. 69-72
`(1987).
`
`Vitetta et al., "Redesigning nature's poisons to create anti(cid:173)
`tumor reagents" Science 238:1098-1104 (1987).
`Vollmar et al., ''Toxicity of Ligand and Antibody-Directed
`Ricin A-Chain Conjugates Recognizing the Epidermal
`J. Cellular Physiology
`Growth Factor Receptor"
`131:418-425 (1987).
`Yamamoto et al., "Similarity of protein encoded by the
`human c-erb-B-2 gene to epidermal growth factor recep(cid:173)
`tor'' Nature 319:230-34 (1986).
`McKenzie et al., "Generation and characterization of mono(cid:173)
`clonal antibodies specific for the human neu oncogene
`product, p185" Oncogene 4:543-548 (1989).
`Muller et al., "Single-Step Induction of Mammary Adeno(cid:173)
`carcinoma in Transgenic Mice Bearing the Activated c-neu
`Oncogene" Cell 54:105-115 (1988).
`Myers et al., "Biological Effects ofMonoclonalAntireceptor
`Antibodies Reactive with neu Oncogene Product, p185""u"
`Methods in Enzymology 198:277-290.
`Drebin et al., "Monoclonal Antibodies Specific for the neu
`Oncogene Product Directly Mediate Anti-tumor Effects In
`Vivo" Oncogene 2 (4):387-394 (1988).
`Hudziak et al., "Amplified Expression of the HER2/ERBB2
`Oncogene Induces Resistance to Tumor Necrosis Factor a in
`NIH 3T3 Cells" Proc. Natl. Acad. Sci. USA 85:5102-5106.
`Aboud-Pirak et al., "Efficacy of Antibodies to Epidermal
`Growth Factor Receptor Against KB Carcinoma In Vitro and
`in Nude Mice" Journal of the National Cancer Institute
`80(20):1605-1611 (Dec. 21, 1988).
`Ballet et al., "Evaluation of a Nude Mouse-Human Tumor
`Panel as a Predictive Secondary Screen for Cancer Chemo(cid:173)
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`(1979).
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`Against an Oncogene--encoded Product Using a Vaccine
`Virus Vector" Proc. Natl. Acad. Sci. USA 84:6854-6858
`(Oct 1987).
`Bucholtz, J.D., ''Radiolabeled Antibody Therapy" Semin.
`Oncol. Nurs. (abstract only) 3(1):67-73 (1987).
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`domains of the neu oncogene--encoded p185 molecule exert
`synergistic anti-tumor effects in vivo" Oncogene 2:273-277
`(1988).
`Hancock et al., "A Monoclonal Antibody Against the
`c--erbB-2 Protein Enhances the Cytotoxicity of cis-Diam(cid:173)
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`Tumor Cell Lines" Cancer Research 51:4575-4580 (Sep. 1,
`1991).
`Masuko et al., "A murine Monoclonal Antibody That Rec(cid:173)
`ognizes an Extracellular Domain of the Human c--erB-2
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`Structure, Expression and Homology to Lymphotoxin"
`Nature 312:724-729 (1984).
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`Reactivity of Monoclonal Antibodies with Recombinant
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`King, C.R. DHHS Patent Application 6/836.414. National
`Technical Information Service, Springfield, VA. 22161 Pub.
`86-197928.
`
`PFIZER EX. 1096
`Page 2
`
`

`

`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 1 of 6
`
`5,677,171
`
`> 0...
`
`0::
`>-
`I-
`::::::! 100
`m
`<(
`>
`1-z
`UJ u
`0::
`UJ a..
`UJ >
`i=
`<(
`_J
`LLI
`0::
`
`100
`
`_J
`0
`0::
`1-z
`0
`u
`LL
`0
`1-z
`I.1.J u
`0::
`I.1.J a..
`~ 40
`<(
`J--
`0...
`:::::>
`L
`L) 20
`..-
`If)
`
`~
`
`Fig .1 a
`
`• NIH 3T3 neo/dhf r
`o HER2-3
`o HER2-3 200
`6 HER2-3400
`X HER2-3aoo
`
`1
`
`10
`
`100
`TNf-C(( U/ml)
`
`1000
`
`10,000
`
`Fig.1 b
`
`• NIH 3T3 neo /dhfr
`X HER 2-3800
`0 NIH 3T3 neo /dhfr H TR
`
`2:1
`1:1
`0·5:1
`0·25:1
`EFFECTOR: TARGET CELL RATIO
`
`4:1
`
`PFIZER EX. 1096
`Page 3
`
`

`

`Fig.2
`
`• NH 3T3 neo /dhtr
`• HER 2-3800
`
`10000
`
`E
`Cl.
`u 8000
`0 z
`:::::>
`0 co
`LJ 6000
`
`LL
`LJ
`lLJ
`CL
`(/)
`~
`I
`LL z
`1-
`I
`........
`1..11
`N ...-
`
`10000
`
`8000
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`X
`
`5 6000
`co
`:::L
`:::::> z
`
`_J
`_I
`
`tJ 4000
`
`2000
`
`Fig.3
`INHIBITION OF SKBR 3 GROWTH BY ANTI-HER
`-2 MABS
`
`0o 0·001 0·01 0·1 1 10 100 1000
`[ TNF-cx] nM
`
`OL----_.J
`
`MAb Supns.
`MAB SUPERNATANTS
`
`1111 myl.supn
`t2J 4(8
`ITI'T9 4 05
`lllillllii
`D 3E8
`D 3H4
`
`~ • 00.
`• ;p
`i
`
`0 p.
`
`.......
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`.......
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`
`PFIZER EX. 1096
`Page 4
`
`

`

`I
`
`0
`
`~
`
`13000
`12000 ' .
`11000~ \
`~ 10000
`~ 9000
`8000
`z
`
`g 7000 ~
`6ooo I
`
`5000.
`
`I
`
`4000
`I
`
`Fig. 4
`
`--------
`
`_.,-
`
`ANTI- HEPATITIS B MAb
`
`\
`---------.
`
`ANTI-HERZ MAb 405
`
`--
`
`0·5
`
`1-0
`
`1· 5
`2·0
`ANTIBODY CONCENTRATION
`}Jg I ml
`
`2·5
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`
`d
`• 00
`•
`~
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`lo-ol.
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`
`PFIZER EX. 1096
`Page 5
`
`

`

`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 4 of 6
`
`5,677,171
`
`Fig.Sa
`
`>
`0... e.: 100
`>-
`j -
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`
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`
`1000
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`5·0
`]JQ/ml Ab
`
`Fig. Sb
`
`>
`0...
`~ 100
`>-
`I-
`___!
`
`60
`
`£l) 80
`4:
`>
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`J.lQfml Ab
`5·0
`
`PFIZER EX. 1096
`Page 6
`
`

`

`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 5 of 6
`
`5,677,171
`
`Fig.6a
`
`>
`0..
`~ 100
`>-
`t-
`_j
`
`(I)
`
`60
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`<! 80
`>
`1-z
`w
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`
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`100
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`1000
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`5·0 ~g/ml Ab
`
`Fig.6b
`
`>
`0..
`~ 100
`>-
`t-
`_ j
`m 80
`<!
`>
`1-z 70
`
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`u
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`100
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`5·0 ~g/ml Ab
`
`PFIZER EX. 1096
`Page 7
`
`

`

`>
`CL
`~ 100
`>-r-
`co 80
`>
`t-z
`w
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`
`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 6 of 6
`
`5,677,171
`
`_J
`
`<(
`
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`
`~'
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`
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`5·0 pg/mlMAb405
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`~ 100
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`
`PFIZER EX. 1096
`Page 8
`
`

`

`5,677,171
`
`1
`MONOCLONAL ANTffiODIES DIRECTED
`TO THE BER2 RECEPTOR
`
`Thls application is a continuation of Ser. No. 07/977,453,
`filed Nov. 18, 1992, now abandoned, which is a continuation
`of Ser. No. 07/147,461, filed Jan. 25, 1988, now abandoned,
`which is a continuation-in-part of Ser. No. 07/143,912, filed
`Jan. 12, 1988, now abandoned, which applications are
`incorporated herein by reference and to which applications
`priority is claimed under 35 U.S.C. § 120.
`
`FIELD OF THE INVENTION
`
`Thls invention is in the fields of immunology and cancer
`diagnosis and therapy. More particularly it concerns anti(cid:173)
`bodies specifically binding growth factor receptors, hybri(cid:173)
`domas that produce these antibodies, immunochemicals
`made from the antibodies, and diagnostic methods that use
`the antibodies. The invention also relates to the use of the
`antibodies alone or in combination with cytotoxic factor(s)
`in therapeutic methods. Also encompassed by the invention
`is an assay for tyrosine kinases that are involved in tumori(cid:173)
`genesis.
`
`BACKGROUND OF THE INVENTION
`
`Macrophages are one of the effector cell types that play an
`important role in immunosurveillance against neoplastic
`growth in vivo. In vitro, cell-mediated cytotoxicity requires
`selective binding between activated macrophages and target
`cells as well as the concomitant release of cytotoxic factors.
`Some of the cytotoxic factors secreted by activated mac(cid:173)
`rophages include reactive oxygen species such as the super(cid:173)
`oxide anion and hydrogen peroxide, arginase, interleukin 1,
`and tumor necrosis factor-a (TNF-a). Acquired resistance to
`the toxic effects of these factors by tumor cells could be one
`mechanism which leads to the onset and spread of tumor
`formation in vivo.
`The observation that TNF-a can act as a potent effector of
`the macrophage-mediated antitumor response provides a
`rationale for its use in further studies on the regulation of 40
`tumorigenesis in vivo and tumor cell growth in vitro. The
`genes encoding TNF-a and TNF-J3, a structurally related
`cytotoxic protein formerly known as lyrnphotoxin, have
`been cloned and the corresponding proteins expressed in
`Escherichia coil. These proteins display an array of biologi- 4s
`cal activities, including induction of hemorrhagic necrosis of
`Meth A sarcomas in vivo, inhibition of the growth of certain
`tumor cells in vitro, synergistic enhancement of the in vitro
`anticellular effects of IFN-y, activation of human polymor(cid:173)
`phonuclear neutrophil functions, and inhibition of lipid
`biosynthesis. Recently, rHuTNF-a was shown to augment
`the growth of normal diploid fibroblasts in vitro. The diver(cid:173)
`gent proliferative responses in the presence of rHuTNF-a
`are sometimes related to variations in TNF binding.
`Growth factors and their receptors are involved in the
`regulation of cell proliferation and they also appear to play
`a key role in oncogenesis. Of the known proto-oncogenes,
`three are related to a growth factor or a growth factor
`receptor. These genes include c-sis, which is homologous to
`the transforming gene of the simian sarcoma virus and is the
`S chain of platelet-derived growth factor (PDGF); c-fms,
`which is homologous to the transforming gene of the feline
`sarcoma virus and is closely related to the macrophage
`colony-stimulating factor receptor (CSF-1R); and c-erbB,
`which encodes the EGFreceptor (EGFR) and is homologous
`to the transforming gene of the arian erythroblastosis virus
`(v-erbB). The two receptor-related proto-oncogenes, c-fms
`
`2
`and c-erbB, are members of the tyrosine-specific protein
`kinase family to which many proto-oncogenes belong.
`Recently, a novel transforming gene was identified as a
`result of transfection studies with DNA from chemically
`5 induced rat neuroblastomas. Thls gene, called neu, was
`shown to be related to, but distinct from, the c-erbB proto(cid:173)
`oncogene. By means of v-erbB and human EGFR as probes
`to screen human genomic and complementary DNA (eDNA)
`libraries, two other groups independently isolated human
`10 erbB-related genes that they called HER2 and c-erbB-2
`respectively. Subsequent sequence analysis and chromo(cid:173)
`somal mapping studies revealed that c-erbB-2, and HER2
`are species variants of neu. A fourth group, also using v-erbB
`as a probe, identified the same gene in a mammary carci-
`15 nomacellline,MAC 117, whereitwasfound to be amplified
`five- to ten-fold.
`Thls gene, which will be referred to herein as HER2,
`encodes a new member of the tyrosine kinase family; and is
`closely related to, but distinct from, the EGFR gene as
`20 reported by Coussens et al., Science 230, 1132 (1985).
`HER2 differs from EGFR in that it is found on band q21 of
`chromosome 17, as compared to band pll-p13 of chromo(cid:173)
`some 7, where the EGFR gene is located. Also, the HER2
`gene generates a messenger RNA (mRNA) of 4.8 kb, which
`25 differs from the 5.8- and 10-.kb transcripts for the EGFR
`gene. Finally, the protein encoded by the HER2 gene is
`185,000 daltons, as compared to the 170,000-dalton protein
`encoded by the EGFR gene. Conversely, on the basis of
`sequence data, HER2 is more closely related to the EGFR
`30 gene than to other members of the tyrosine kinase family.
`Like the EGFR protein, the HER2 protein (p185) has an
`extracellular domain, a transmembrane domain that includes
`two cysteine-rich repeat clusters, and an intracellular kinase
`domain, indicating that it is likely to be a cellular receptor
`35 for an as yet unidentified ligand. HER2 p185 is referred to
`as p185 or the HER2 receptor herein.
`Southern analysis of primary human tumors and estab-
`lished tumor-derived cell lines revealed amplification and in
`some cases rearrangement of the EGF receptor gene. Ampli(cid:173)
`fication was particularly apparent in squamous carcinomas
`and glioblastomas. The HER2 gene was also found to be
`amplified in a human salivary gland adenocarcinoma, a renal
`adenocarcinoma, a mammary gland carcinoma, and a gastric
`cancer cell line. Recently, Slamon et al., Science 235, 177
`(1987) demonstrated that about 30% of primary human
`breast carcinoma tumors contained an amplified HER2 gene.
`Although a few sequence rearrangements were detected, in
`most tumors there were no obvious differences between
`amplified and normal HER2 genes. Furthermore, amplifica-
`50 tion of the HER2 gene correlated significantly with the
`negative prognosis of the disease and the probability of
`relapse.
`To investigate the significance of the correlation between
`overexpression and cellular transformation as it has been
`55 observed for proto-oncogenes c-mos and N-myc, a HER2
`expression vector and a selection scheme that permitted
`sequence amplification after transfection of mouse Nlli 3T3
`cells was employed by Hudziak et al., Proc. Nat[. Acad. Sci.
`(USA) 84, 7159 ( 1987). Amplification of the unaltered
`60 HER2 gene in Nlli3T3 cells lead to overexpression ofp185
`as well as cellular transformation and tumor formation in
`athymic mice.
`The effects of antibodies specifically binding growth
`factors or growth factor receptors has been studied.
`65 Examples are discussed below.
`Rosenthal et al., Cell46, 301 (1986) introduced a human
`TGF-a eDNA expression vector into established non-
`
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`5,677,171
`
`4
`It is a further object of the invention To provide improved
`methods of tumor therapy.
`It is a further object of the invention to provide a method
`of inhibiting the growth of tumor cells which overexpress a
`5 growth factor receptor and/or growth factor.
`It is a further object of the invention to provide a method
`for treating a tumor by treatment of the tumor cells with
`antibodies capable of inhibiting growth factor receptor
`function, and with cytotoxic factors such as tumor necrosis
`factor.
`A still further object of the invention is to provide an assay
`for tyrosine kinases that may have a role in tumorigenesis.
`Other objects, features and characteristics of the present
`15 invention will become apparent upon consideration of the
`following description and the appended claims.
`
`w
`
`3
`transformed rat fibroblast cells. Synthesis and secretion of
`TGF-a by these cells resulted in loss of anchorage(cid:173)
`dependent growth and induced tumor formation in nude
`mice. Anti-human TGF-a monoclonal antibodies prevented
`the rat cells from forming colonies in soft agar, i.e. loss of
`anchorage dependence. Gill et al. in J. Bioi. Chern. 259,
`7755 (1984) disclose monoclonal antibodies specific for
`EGF receptor which were inhibitors of EGF binding and
`antagonists of EGF-stimulated tyrosine protein kinase activ-
`~
`Drebin et al. in Cell 41, 695 (1985) demonstrated that
`exposure of a neu-oncogene-transformed Nlli 3T3 cell to
`monoclonal antibodies reactive with the neu gene product,
`cause the neu-transformed NIH 3T3 cell to revert to a
`non-transformed phenotype as determined by anchorage
`independent growth. Drebin et al. in Proc. Natl. Acad. Sci.
`83, 9129 (1986) demonstrated that in vivo treatment with a
`monoclonal antibody (IgG2a isotype) specifically binding
`the protein encoded by the neu oncogene significantly
`inhibited the tumorigenic growth of neu-transformed Nlli
`3T3 cells implanted into nude mice.
`Akiyama et al. in Science 232, 1644 (1986) raised anti(cid:173)
`bodies against a synthetic peptide corresponding to 14
`amino acid residues at the carboxy-terminus of the protein
`deduced from the c-erbB-2 (HER2) nucleotide sequence.
`Growth factors have been reported to interact in both a
`synergistic and an antagonistic manner. For example, TGF-a
`and TGF-~ synergistically enhance the growth of NRK-49F
`fibroblasts, whereas PDGF down regulates EGF receptor
`function on 3T3 cells. A variety of transformed cells secrete 30
`factors which are believed to stimulate growth by an auto(cid:173)
`crine mechanism. Sugarman et al., Cancer Res. 47, 780
`(1987) demonstrated that under certain conditions, growth
`factors can block the antiproliferative effects of TNF-a on
`sensitive tumor cells. Specifically, epidermal growth factor 35
`(EGF). and recombinant human transforming growth
`factor-a (rHuTGF-a) were shown to interfere with the in
`vitro antiproliferative effects of recombinant human tumor
`necrosis factor-a (rHuTNF-a) and -~ on a human cervical
`carcinoma cell line, :ME-180. The inhibitory effect could be 40
`observed at EGF or rHuTGF-a concentrations of OJ to 100
`ng/ml, and was maximal between 1 and 10 ng/ml. This
`response was apparently not due to down regulation of the
`TNF receptor or to alteration of the affinity ofTNF-a for its
`receptor. Since the antiproliferative effect of recombinant 45
`human interferon-y was not significantly affected by the
`presence of EGF or rHuTGF-a, the inhibition was specific
`for recombinant TNFs and was not due solely to enhanced
`proliferation induced by the growth factors. Neither growth
`factor had a substantial protective effect on the synergistic 50
`cytotoxicity observed when tumor cells were exposed simul(cid:173)
`taneously to rHuTNF-a and recombinant human interferon-
`'¥· TGF-o can also interfere with the antiproliferative effects
`of rHuTNF-a in vitro. At concentrations of less than 1
`ng/ml, TGF-~ significantly antagonized the cytotoxic effects 55
`of rHuTNF-a on Nlli 3T3 fibroblasts. Since EGF, platelet(cid:173)
`derived growth factor, and TGF-~ all enhanced Nlli 3T3 cell
`proliferation, but only TGF-~ interfered with rHuTNF-a
`cytotoxicity, the protective effects ofTGF-~ were not related
`in a simple manner to enhanced cell proliferation. 60
`rHuTGF-a and TGF-~ did not have a significant protective
`effect against rHuTNF-a-mediated cytotoxicity on two other
`tumor cell lines, BT-20 and L-929 cells.
`It is an object of the subject invention to provide anti(cid:173)
`bodies capable of inhibiting growth factor receptor function. 65
`It is a further object of the invention to provide an
`improved assay for the HER2 receptor.
`
`20
`
`SUMMARY OF THE INVENTION
`The subject invention relates to monoclonal antibodies
`specifically binding the external domain of the HER2 recep(cid:173)
`tor. The invention also relates to an assay for the HER2
`receptor comprising exposing cells to antibodies specifically
`binding the extracellular domain of the HER2 receptor, and
`determining the extent of binding of said antibodies to said
`25 cells. Another embodiment of the invention relates to a
`method of inhibiting growth of tumor cells by administering
`to a patient a therapeutically effective amount of antibodies
`capable of inhibiting the HER2 receptor function. A further
`embodiment of the invention relates to administering a
`therapeutically effective amount of antibodies capable of
`inhibiting growth factor receptor function, and a therapeu-
`tically effective amount of a cytotoxic factor. A still further
`embodiment of the invention is an assay for tyrosine kinases
`that may have a role in tumorigenesis comprising exposing
`cells suspected to be TNF-a resistant to TNF-a, isolating
`those cell which are TNF-a resistant, screening the isolated
`cells for increased tyrosine kinase activity, and isolating
`receptors and other proteins having increased tyrosine
`kinase activity.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. la shows TNF-a resistance of NIH 3T3 cells
`expressing various levels of HER2 p185. FIG. lb shows
`macrophage cytotoxicity assays for NIH 3T3 cells express(cid:173)
`ing various levels of HER2 p185.
`FIG. 2 demonstrates the level of TNF-a binding for a
`control cell line (NTII 3T3 neo/dhfr) and for a cell line
`overexpressing HER2 p185 (HER2-3800).
`FIG. 3 shows inhibition of SK BR3 cell growth by
`anti-HER2 monoclonal antibodies.
`FIG. 4 is a dose response curve comparing the effect of an
`irrelevant monoclonal antibody (anti-HBV) and the effect of
`monoclonal antibody 4D5 (anti-HER2) on the growth of SK
`BR3 cells in serum.
`FIGS. Sa, Sb and 6a show percent viability of SK BR3
`cells as a function of increasing TNF-a concentration and
`anti-HER2 p185 monoclonal antibody concentration. Each
`Figure shows the results for a different anti-HER2 p185
`monoclonal antibody. FIG. 6b is a control using an irrelevant
`monoclonal antibody. In FIGS. Sa, Sb, 6a and 6b, e-e
`represents TNF-a alone; ._ . represents antibody (Ab)
`alone; o-o represents 100 Uml TNF-a and Mb; D-0
`represents 1000 U/ml TNF-a and Mb; and~-~ represents
`10,000 U/ml TNF-a and Mb.
`FIG. 7 shoes percent viability ofMDA-MB-175-VII cells
`as a function of increasing TNF-a concentration and anti-
`
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`5,677,171
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`5
`HER2 pl85 monoclonal antibody concentration. In FIG. 7,
`• - • represents TNF-a alone; -
`represents antibody
`(Ab) alone; o-o represents 100 U/ml TNF-a and Mb;
`D-0 represents 1000 U/ml TNF-a and Mb; and .11-d
`represents 10,000 U/ml TNF-a and Mb.
`FIG. 8 shows percent viability of Nlli 3T3 cells overex(cid:173)
`pressing HER2 pl85 as a function of increasing TNF-a
`concentration and anti-HER2 pl85 monoclonal antibody
`concentration. In FIG. 8, • - • represents TNF-a alone;
`-represents antibody (Ab) alone; o-o represents 100
`U/ml TNF-a and Mb; D-D represents 1000 U/ml TNF-a
`and Mb; and d-d represents 10,000 U/ml TNF-a and
`Mb.
`
`6
`complement and/or mediate antibody-dependent cellu(cid:173)
`lar cytotoxicity (ADCC), e.g. IgG2a antibodies;
`(g) The antibodies which bind the receptor or growth
`factor are conjugated to a toxin (immunotoxins);
`5 Advantageously antibodies are selected which greatly
`inhibit the receptor function by binding the steric vicinity of
`the ligand binding site of the receptor (blocking the
`receptor), and/or which bind the growth factor in such a way
`as to prevent (block) the ligand from binding to the receptor.
`10 These antibodies are selected using conventional in vitro
`assays for selecting antibodies which neutralize receptor
`function. Antibodies that act as ligand agonists by mimick(cid:173)
`ing the ligand are discarded by conducting suitable assays as
`will be apparent to those skilled in the art. For certain tumor
`DErAILED DESCRIPTION OF THE
`15 cells, the antibodies inhibit an autocrine growth cycle (i.e.
`INVENTION
`where a cell secretes a growth factor which then binds to a
`A new application of antibodies to inhibit the growth of
`receptor of the same cell). Since some ligands, e.g. TGF-a,
`are found lodged in cell membranes, the antibodies serving
`tumor cells has been discovered. Surprisingly it has been
`a targeting function are directed against the ligand and/or the
`found that by inhibiting growth factor receptor function, e.g.
`the HER2 receptor function, cell growth is inhibited, and the. 20 receptor.
`cells are rendered more susceptible to cytotoxic factors.
`Certain tumor cells secrete growth factors that are
`Thus, for example, breast cancer cells which are refractory
`required for normal cellular growth and division. These
`to TNF-a alone can be made susceptible to TNF-a if the
`growth factors, however, can under some conditions stimu-
`cells are first treated with antibodies which inhibit growth
`late unregulated growth of the tumor cell itself, as well as
`factor receptor function. The increase of susceptibility has 25 adjacent non-tumor cells, and can cause a tumor to form.
`been demonstrated using the HER2 receptor and monoclonal
`Epidermal Growth Factor (EGF) has dramatic stimulatory
`antibodies directed against the HER2 receptor, and tumor
`effects on cell growth. In purified receptor preparations, the
`necrosis factor-a.
`EGF receptor is a protein kinase that is activated by the
`The method of this invention is useful in the therapy of
`binding of EGF. Substrate proteins for this kinase are
`malignant or benign tumors of mammals where the abnor- 30 phosphorylated on tyrosine residues. The receptors for
`mal growth rate of the tumor is dependent upon growth
`insulin, platelet-derived growth factor (PDGF) and oth