111111111111111111
`
`US005677 l 71A
`£11] Patent Number:
`[45] Date of Patent:
`
`5,677,171
`Oct. 14, 1997
`
`United States Patent (19]
`Hudziak et al.
`
`[54] MONOCLONAL ANTIBODIES DIRECTED
`TO THE HER2 RECEPTOR
`
`[75]
`
`Inveotors: Robert M. Budzia.k. Corvallis, Oreg.;
`H. Michael Shepard, Ranch o Saota Fe.
`Calif.; Axel Ullrich, Portola Valley,
`Calif.; Brian M. Fendly, Half Moon
`Bay. Calif.
`
`[73] Assignee: Genentech, Ioc.. South San Francisco,
`Calif.
`.
`
`[21] Appl. No.: 286,303
`
`[22] Filed:
`
`Aug. 5, 1994
`
`Related U.S. Application Data
`
`[63) ContinWllion of Sei: 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 Sec. 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; 4351112.2
`[58) Field of Search .............................. 530/387.7, 388.8,
`530/388.85, 381.l; 435n.23, 240.21. 112.2,
`70.21
`
`[56]
`
`References Cited
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`9/1990 Huang .................................... 424/85.8
`4,968,603 1111990 Slamon et al . .
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`4/1992 Rokugawa et al ..
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`5,401,638
`3/1995 Camey et al . .
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`0118365
`8/1994 European Pat. Off. ........ CUN 15..00
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`85/03357
`811985 WlPO .
`87/07646 1211987 WlPO .
`WO 89/10412 1111989 WIPO .............................. C12Q U68
`
`OTHER PUBLICATIONS
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`
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`Drebin et al.. "Inhibition of tumor growth by a monoclonal
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`Gill et aL, ''Monoclonal Anti-epidermal Growth Factor
`Receptor Antibodies Which are Inhibitors of Epidermal
`Growth Factor Binding and Antagonists of Epidermal
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`nal of Biological Chemistry 259:7755-60 (1984).
`Hudziak et al., "Increased expression of the putative growth
`factor receptor p185HERl causes transformation and tumori(cid:173)
`genesis of NIH 3T3 cells" Proc. Natl Acad. Sci.
`84:7159-7163 (1987).
`Hudziak et al., "pl85HER2 Monoclonal Antibody Has Anti(cid:173)
`prolifaative Effects In Vitro and Sensitizes Human Breast
`Tumor Cells to Tumo.r Necrosis Factor" M olecular & Cel(cid:173)
`lular Biology 9(3):1165-1172 (1989).
`King et al, "Amplification of a Novel v-c.-bB-Related Gene
`in a Human Mammary Carcinoma" Science 229:974-976
`(1985).
`Kipps et al., "Schemata for the production of monoclonal
`antibody-producing hybridomas" Handbook of Experimen(cid:173)
`tal /mmunol-Ogy. Herzenberg et al. (eds.), Blackwell Sci.
`Chapter 108, pp. 108.1 -108.9 (1986).
`Lupu et aL, "Direct interactioo of a ligand for the erbB2
`oncogene product with the EGF receptor and p l85ut>82
`"
`Science 249:1552-1555 (1990).
`Maosi et al., "In vivo Evaluation of an Aoti-Melanoma
`Antibody, F(ab')2. Labelled with TC-99m" Oncogenes to
`Tunwr Antigens, Geraldo et al, eds., Elsevier Sci. Publ.
`(1985).
`
`(List cootioued on oex.t page.)
`
`Primary Examiner-Lila Feisee
`Assistant Examiner-Geelha P. Bansal
`Attorney, Agent, or Finn-Wendy M. Lee
`[57]
`
`ABSTRACT
`
`M onoclonal 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 tumOi" characterized by amplified
`expression of HER2.
`
`39 Claims, 6 Drawing Sheets
`
`1 of 19
`
`BI Exhibit 1096
`
`

`

`5,677,171
`Page2
`
`OTHER PUBLICM'IONS
`Masui et al., "Mechanism of Antitwnor Activity in Mice for
`Anti-Epidermal Growth Factor Receptor Monoclonal Anti(cid:173)
`bodies with Different
`lsotypes" Cancer Research
`46;5592-5598 (1986).
`Morrison, 'Transfectomas Provide Novel Orimeric Anti(cid:173)
`bodies" Science 229:1202-1207 (1985).
`Pad.by et al., "Identification of a Pbosphoprotein Specifically
`Induced by the Transforming DNA of Rat Neuroblastomas"
`Cell 28:865--871 (1982).
`Roded: et al.. '1nteractioos between growth factor receptors
`and corresponding monoclonal antibodies in human tumors"
`J. Cellular Biochem. 35(4):315-320 (1987).
`Roded: 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-ex cDNA Results in Transfor(cid:173)
`mation" Cell 46:301-309 (1986).
`Sato et al., "Biological Effects in Vitro of Monoclonal
`Antibodies to Homan Epidermal Growth Factor Receptors"
`Mol. Biol Med. 1:511-529 (1983).
`Schechter et al., 'The oeu Gene: Ao erbB-Homologous
`Gene Distinct from and Unllked to the Gene Encoding the
`EGF Receptor" Science 229:976-978 (1985).
`Schlom et al., "Basic Principles and Applications of Mono(cid:173)
`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-abB-rclated protooocogene c-erbB-2.
`is distinct from the c--erb-B-1/cpidermal 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/oeu
`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)
`lifcrative Activity of Tumor Necrosis Factor" Cancer
`Research 47:780-786 (1987).
`Sugarman et al.. "Recombinant Human 'Tumor Necrosis
`Factor-ex: Effects on Proliferation of Normal and Trans(cid:173)
`formed Cells in Vitro" Science 230:943-945 (1985).
`Takahashi et al., ''Radioimmunodetection of Human Glioma
`Xenografts by Monoclonal Antibody to Epidermal Growth
`Factor Receptoc'' Cancer Reserch 47:3847-3850 (1987).
`Urban et al., 'Tumor necrosis factor: A p otent 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 nen ( c-erbB-2)
`Oncogene in Human Mammary Tumors Is Relatively Fro(cid:173)
`queot and Is Often Accompanied by Amplification of the
`Linked c--erbA Oncogene" Molecular &: Cellular Biology
`7(5):2019-2023 (1987).
`Venter et al.. "Overcxpressioo of the c-erbB-2 Oncoprotein
`in Human Breast Carcinomas: Immuoohistological Assess(cid:173)
`ment Correlates with Gene Amplification" U:mcet 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., 'Toxici.ty of Ligand and A ntibody-Directed
`Ricin A-Chain Conjugates Recognizing the Epidermal
`Growth Factor Receptor"
`J. Cellular Physiology
`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 of Monoclonal Antireceptor
`Antibodies Reactive with oeu Oncogene Product, p185"'"'"
`Methods in Enzymology 198:277-290.
`Drebio et al.. "Monoclonal Antibodjcs 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 ex in
`NIH 3T3 Cells" Proc. Natl. Acad. Sci. USA 85:5102-5106.
`Aboud-Pirak et al., "Efficacy of Antibodies to Epidennal
`Growth Factor Receptor Against KB Carcinoma In Vitro and
`lo 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)
`therapeutic Agenst" J. Natl. Cane. Inst 63(5):1185-1188
`(1979).
`Bernards et al., "Effective Tumor lmmunothcrapy Directed
`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).
`Drebin et al., "Monoclonal antibodies reactive with distinct
`domains of the neu oncogene-encoded pl85 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)
`minedichloroplatinum Against Human Breast and Ovarian
`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
`Protooncogene Product" Jpn J. Cancer Res. 80:10-14 (Jan.
`1989).
`Peonica et al., "Hum.an Tumour Necrosis Factor: Precursor
`Structure, Expression and Homology to Lympbotoxin"
`Nature 312:724-729 (1984).
`Ring et al., "Identity of BCA200 and c-erB-2 Indicated by
`Reactivity of Monoclonal Antibodies with Recombinant
`c-erbB-2" Moleculllr Immunology 28(8):915-917 (1991).
`King, C.R. DHHS Patent Application 61836.414. National
`Technical Information Se.rvice, Springfield, VA 22161 Pub.
`86-197928.
`
`2 of 19
`
`BI Exhibit 1096
`
`

`

`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 1of6
`
`5,677,171
`
`140
`
`Fig .1 a
`
`> CL
`
`0::
`>-
`I-
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`a::l
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`o HER2-3
`o HER2-3 200
`6 HER2-3400
`x HER2-3soo
`
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`
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`
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`1:1
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`EFFECTOR :TARGET CELL RATIO
`
`4:1
`
`3 of 19
`
`BI Exhibit 1096
`
`

`

`r
`
`Fig.2
`
`10000 I
`
`• NH 3T3 neo /dhtr
`I • HER 2-3aoo
`
`-
`
`Fig .3
`INHIBITION OF SKBR 3 GROWTH BY ANTI-HER
`- 2 MABS
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`4 of 19
`
`BI Exhibit 1096
`
`

`

`14000r
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`5 of 19
`
`BI Exhibit 1096
`
`

`

`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 4 of 6
`
`5,677,171
`
`Fig.Sa
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`
`6 of 19
`
`BI Exhibit 1096
`
`

`

`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 5 of 6
`
`5,677,171
`
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`
`7 of 19
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`BI Exhibit 1096
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`

`

`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 6 of 6
`
`5,677,171
`
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`
`8 of 19
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`BI Exhibit 1096
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`

`

`1
`MONOCWNAL ANTIBODIES DIRECTED
`TO THE HER2 RECEPTOR
`
`5,677,171
`
`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
`s induced rat neuroblastomas. This 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 ( cDNA)
`hbraries, 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 noma cell line, MAC 117. where it was fou nd to be amplified
`Ii ve- to ten-fold.
`This gene, which will be referred to herein as HER2,
`encodes a new member of the tyros.ine 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-pl3 of chromo(cid:173)
`some 7. where the EGFR gene is located. Also, the HER2
`gene generates a messenger RNA (rnRNA) 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 cysteioe-rich repeat clusters. and an intracellular kinase
`domain. indicating that it is likely to be a cellular receptor
`for an as yet unidentified ligand. HER2 p185 is referred to
`as pl85 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-
`so tion of the llER2 gene correlated significantly with the
`negative prognosis of the disease and the probability of
`relapse.
`To .investigate the significance of lhe correlation between
`overexpression and cellular transformation as it has been
`ss observed for proto-oncogenes o-mos and N-myc, a HER2
`expression vector and a selection scheme that permitted
`sequence amplification after transfection of mouse Nm: 3T3
`cells was employed by Hudz.iak et al., Proc. Natl Acad. Sci.
`(USA) 84, 7159 (1987). Amplification of the unaltered
`60 HBR2 gene in ND:l 3T3 cells lead to ovcre:xpression of p185
`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.
`6S Examples are discussed below.
`Rosenthal et al., Cell 46. 301 (1986) introduced a human
`TGF-a cDNA expression vector into established non-
`
`Tiris application is a continuation of Ser. No. 07 /<J'J7 ,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. 07/143,9U. filed
`Jan. 12, 1988, now abandoned, which applications are
`incoxporated herein by reference and to which applications
`)X:iority is claimed under 35 U.S.C. § 120.
`
`FIELD OF THE INVENTION
`This 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
`Maaopbages are one of the effector cell types that play an
`important role in iromunosurveillance 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 (fNF-a). Acquiredresistance to
`the toxic effects of these factors by tumor cells could be one 35
`mechanism which leads to the onset and spread of turner
`formation in vivo.
`The observation thatTNF-a can act as a potent effector of
`the maaophage-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-~, a structurally related
`cytotoxic protein formerly known as lymphotoxin, have
`been cloned and the corresponding proteins expressed in
`Escherichia coil. These proteins display an array of biologi- 45
`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 JFN-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 nonnal 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 (POOF); c-fms.
`which is homologous to the transforming gene of the feline
`sarcoma virus and is closely related to the maaophage
`colony-stimulating factor receptor (CSF-lR); and c-erbB.
`which encodes the EGF receptor (EGFR) and is homologous
`to the transforming gene of the arian erythroblastosis virus
`(v-erbB). The two receptor-related proto-oncogenes. c-fms
`
`9 of 19
`
`BI Exhibit 1096
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`15
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`20
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`25
`
`3
`transformed rat fibroblast cells. Synthesis and secretion of
`TGF-cx by these cells resulted in loss of anchorage(cid:173)
`dependent growth and induced tumor formation in nude
`mice. Anti-human TGF-cx monoclonal antibodies prevented
`the rat cells from forming colonies in soft agar. i.e. loss of 5
`anchorage dependence. Gill et al. in J. Biol. Chem. 259,
`7755 (1984) disclose monoclonal antibodies speciftc for
`EGF receptor which were inhibitors of EGF binding and
`antagonists ofEGF-stimulated tyrosine protein kinase activ-
`~
`Drebin et al. in Cell 41, 695 (1985) demonstrated that
`exposure of a neu-oncogene-transfooned NIH 3T3 cell to
`monoclonal antibodies reactive with the neu gene product.
`cause the neu-transfonned Nill 3T3 cell to revert to a
`non-transformed phenotype as determined by anchorage
`independent growth. Drebin et al. in Proc. Nall. 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-transfonned Nill
`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-o:
`and TGF-~ synergistically enhance the growth ofNRK-49F
`fibroblasts, whereas POOF 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)
`aine mechanism. Sugarman et al., Cancer Res. 47. 780
`(1987) demonstrated that under certain conditions, growth
`factors can block the antiproliferative effects of TNF-cx on
`sensitive tumor cells. Specifically. epidermal growth factor 35
`(EGF). and recombinant human transforming growth
`factor-a. (rHuTGF-o:) were shown to inteifere with the in
`vitro antiproliferative effects of recombinant human tumor
`necrosis factor-ex (rHuTNF-o:) and -~ on a human cervical
`carcinoma cell line. ME-180. The inhibitory effect could be 40
`observed at EGF or rHuTGF-cx concentrations of 0.1 to 100
`ng/ml, and was maximal between 1 and 10 ng/ml. This
`response was apparently not due to down regulation of the
`1NF receptor or to alteration of the affinity of1NF-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-cx, the inhibition was specific
`for recombinant 1NFs 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 rHu1NF-cx and recombinant human int.erfcron-
`y. TGF-o can also interfere with the antiproliferative effects
`of rHu1NF-cx in vitro. At concentrations of less than 1
`ng/ml, TGF-~ significantly antagonized the cytotoxic effects 55
`of r HuTNF-a. on Nill 3T3 fibroblasts. Since EGF, platelet(cid:173)
`derived growth factor. and TGF-~ all enhanced NIB 3T3 cell
`proliferation, but only TGF-~ interfered with rHuTNF-cx
`cytotoxicity, the protective effects ofTGF-~ were not related
`in a simple manner to enhanced cell proliferation.
`rHuTGF-cx and TGP-~ did not have a significant protective
`effect against rHu1NF-cx-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 fu nction.
`It is a further object of the invention to provide an
`improved assay for the HER2 receptor.
`
`5,677,171
`
`w
`
`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
`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
`invention will become apparent upon consideration of the
`following description and the appended claims.
`
`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 antJ1x><lies specifically
`binding the extracellular domain of the llER2 receptor, and
`determining the extent of binding of said antibodies to said
`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 effe.ctive amount of antibodies capable of
`inhibiting growth factor receptor function, and a therapeu(cid:173)
`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-cx resistant to TNF-o:, isolating
`those cell which are TNF-cx 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 DRAWJNGS
`FIG. l a shows 1NF-cx resistance of Nill 3T3 cells
`expressing various levels of HER2 plS.5. 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 (Nill 3T3 neoldhfr) and for a cell line
`ovcrexpressing HER2 p185 (HER2-3eoo).
`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 serwn.
`FIGS. Sa. Sb and 6a show percent viability of SK BR3
`cells as a function of increasing 1NF-cx concentration and
`anti-HER2 p185 monoclonal antibody concentration. Each
`Figure shows the results for a different anti-HER2 p185
`60 monoclonal antibody. FIG. lib is a control using an irrelevant
`monoclonal antibody. In FIGS. Sa, Sb. 6a and lib. e-e
`represents antibody (Ab)
`represents TNF-o: alone; -
`alone; o-o represents 100 Uml 1NF-<X and Mb; 0-0
`represents 1000 U/ml TNF-cx and Mb; and 6-6 represents
`6S 10.000 U/ml TNF-o: and Mb.
`FIG. 7 shoes percent viability ofMDA-MB-175-VII cells
`as a function of increasing 1NF-cx concentration and anti-
`
`10 of 19
`
`BI Exhibit 1096
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`

`

`5,677,171
`
`5
`HER2 pl 85 monoclonal antil>ody concentration. In flG. 7,
`e-e rei--esents TNF-a. alone; ._ . represents antibody
`(Ab) alone; o-o represents 100 U/ml 1NP-a and Mb;
`0-0 represents 1000 U/ml TNF-a and M b; and 11-11
`rei--esents 10.000 U/ml TNF-a and Mb.
`FIG. 8 shows percent viability of Nm 3T3 cells overex(cid:173)
`pressing HER2 p185 as a function of increasing TNF-a
`concentration and anti-RER2 pl85 monoclonal antibody
`concentration. In flG. 8, • - • represents TNF-a alone;
`._ . rei--esents antibody (Ab) alone; o-o represents 100
`U/ml TNF-a and Mb; 0-0 represents 1000 U/ml TNF-<X
`and Mb; and 6-6 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 a.ntibodies;
`(g) The antibodies which bind the receptor or growth
`factor are conjugated to a toxin (immunotoxins);
`s 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 th.at 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. Por certain tumor
`DETAILED DESCRIPTION OF THE
`is cells, the antibodies inhibit an autocrine growth cycle (i.e.
`INVENTION
`where a cell seaetes 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,
`tumor cells bas been discovered. Surprisingly it has been
`are found lodged in cell membranes, the antibodies serving
`found that by inhibiting growth factor receptor function, e.g.
`a targeting function are directed against the ligand and/or the
`the RER2 receptor function. cell growth is inlul>ited. 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-
`ceUs are first treated with antibodies which inhibit growth
`late unregulated growth of the tumor cell itself. as well as
`factor receptor function. The inaease of susceptibility bas 25 adjacent non-tumor cells. and can cause a tumor to form.
`been demonstrated using the HER2 receptor and monoclonal
`Epidennal Growth Pactor (EGF) has dramatic stimulatory
`antibodies directed against the HER.2 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 EGP. 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 t