United States Patent [191 -
`Hudziak et al.
`
`[54] MONOCLONAL ANTIBODIES 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 US. Application Data
`
`[63] Continuation of Ser. No. 977,453, Nov. 18, 1992, aban
`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.
`
`[51] Int. Cl.6 ....................... .. C07K 16/00; G01N 33/574
`[52] us. (:1. ............................ .. 435124027; 530/388.8;
`530/388.85; 530/387.7; 435/723; 435/172.2
`[58] Field of Search ............................ .. 530/3877, 388.8,
`530/388.85, 381.1; 435/723, 240.27, 172.2,
`70.21
`
`[56]
`
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`[11] Patent Number:
`[45] Date of Patent:
`
`5,677,171
`Oct. 14, 1997
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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. vLee
`[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
`VilIO assays for detecting a tumor characterized by ampli?ed
`expression of HER2.
`
`39 Claims, 6 Drawing Sheets
`
`HOSPIRA EX. 1108
`Page 1
`
`

`

`5,677,171
`Page 2
`
`OTHER PUBLICATIONS
`
`Masui et al., “Mechanism of Antitumor Activity in Mice for
`Anti-Epiderrnal Growth Factor Receptor Monoclonal Anti
`bodies with Different Isotypes” Cancer Research
`46:5592-5598 (1986).
`Monison, ‘Transfectomas Provide Novel Chimeric Anti
`bodies” Science 229zl202-1207 (1985).
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`Induced by the Transforming DNA of Rat Neuroblastomas”
`Cell 28:865-871 (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: Irnmu
`nologically Mediated and Etfector Cell-independent
`Elfects” Cancer Research 47(14)::5692-3696 (1987).
`Rosenthal et al., “Expression in Rat Fibroblasts of a Human
`Transforming Growth Factor-0t cDNA Results in Transfor
`mation” Cell 46:301-309 (1986).
`Sato et al., “Biological Effects in Vitro of Monoclonal
`Antibodies to Human Epidermal Growth Factor Receptors”
`Mol. Biol. Med. 1:511-529 (1983).
`Schechter et al., “The neu Gene: An erbB-Homologous
`Gene Distinct from and Unliked to the Gene Encoding the
`EGF Receptor” Science 229:976-978 (1985).
`Schlom et al., “Basic Principles and Applications of Mono
`clonal Antibodies on the Management of Carcinomas: The
`Richard and Hinda Rosenthal Foundation Award Lecture”
`Cancer Research 46:3225-3238 (1986).
`Semba et al., “A v-erbB-related Protooncogene c-erbB-2,
`is distinct from the c-erb-B-l/epidermal growth factor
`receptor gene and is ampli?ed 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 Ampli?cation of the HER-2/neu
`Oncogene” Science 235:177-182 (1987).
`Sobol et a1. , “Epidermal Growth Factor Receptor Expression
`in Human Lung Carcinoma De?ned by a Monclonal Anti
`body” J. Natl. Cancer Institute 79(3):403-407 (1987).
`Sugarman et al., “Eifects of Growth Factors on the Antipro
`liferative Activity of Tumor Necrosis Factor” Cancer
`Research 47:780-786 (1987).
`Sugarman et al., “Recombinant Human Tumor Necrosis
`Factor-0t: Effects on Proliferation of Normal and Trans
`formed Cells in Vitro” Science 230:943-945 (1985).
`Takahashi et al., “Radioimmunodetection 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., “Ampli?cation of the neu (c-erbB-2)
`Oncogene in Human Mammary Tumors Is Relatively Fre
`quent and Is Often Accompanied by Ampli?cation of the
`Linked c-erbA Oncogene” Molecular & Cellular Biology
`7(5):2019-2023 (1987).
`Venter et al., “Overexpres sion of the c-erbB-2 Oncoprotein
`in Human Breast Carcinomas: Immunohistological Assess
`ment Correlates with Gene Ampli?cation” Lancet pp. 69-72
`(1987).
`
`Vitetta et al., “Redesigning natnre’s poisons to create anti
`tumor reagents” Science 238:1098-1104 (1987).
`Vollmar et al., ‘Toxicity of Ligand and ‘Antibody-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
`tor” Nature 319:230-34 (1986).
`McKenzie et al., “Generation and characterization of mono
`clonal antibodies speci?c for the human neu oncogene
`product, p185” Oncogene 4543-548 (1989).
`Muller et al., “Single-Step Induction of Mammary Adeno
`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 neu Oncogene Product, p185"e"”
`Methods in Enzymology 198 :277-290.
`Drebin et aL, “Monoclonal Antibodies Speci?c for the neu
`Oncogene Product Directly Mediate Anti-tumor Eifects In
`Vivo” Oncogene 2 (4}:387-394 (1988).
`Hudziak et al., “Ampli?ed Expression of the HER2/ERBB2
`Oncogene Induces Resistance to Tumor Necrosis Factor on in
`NIH 3T3 Cells” Proc. Natl. Acad. Sci. USA 85:5102-5106.
`Aboud-Pirak et al., “E?icacy 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
`therapeutic Agenst” J. Natl. Canc. Inst 63(5):1185-1188
`(1979).
`‘
`Bernards et al., “Effective 'Iumor Immunotherapy 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 p185 molecule exert
`synergistic anti-tumor e?ects 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
`minedichloroplatinum Against Human Breast and Ovarian
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`ognizes an Extracellular Domain of the Human c-erB-2
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`1989).
`Pennica et al., “Human Tumour Necrosis Factor: Precursor
`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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`Technical Information Service, Spring?eld, VA. 22161 Pub;
`86-197928.
`
`HOSPIRA EX. 1108
`Page 2
`
`

`

`US. Patent
`
`Oct. 14, 1997
`
`Sheet 1 of 6
`
`5,677,171
`
`Fig.1a
`
`[5—ot
`
`SESSY\
`© HER2-3
`
`® NIH3T3neo/dhfr
`
` RELATIVEPERCENTVIABILITY(RPV)
`
`
`
` 100
`
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`
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`
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`
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`
`as
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`
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`
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`
`1000
`
`@ NIH3T3 neo/dhfr
`
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`
`
`0
`0:25:1
`05:1
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`2:1
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`
`HOSPIRA EX. 1108
`Page 3
`
`HOSPIRA EX. 1108
`Page 3
`
`

`

`US. Patent
`
`Oct. 14, 1997
`
`Sheet 2 of 6
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`5,677,171
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`HOSPIRA EX. 1108
`Page 4
`
`

`

`US. Patent
`
`Oct. 14, 1997
`
`Sheet 3 of 6
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`5,677,171
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`HOSPIRA EX. 1108
`Page 5
`
`

`

`US. Patent
`
`Oct. 14, 1997
`
`Sheet 4 of 6
`
`5,677,171
`
`I
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`
`HOSPIRA EX. 1108
`Page 6
`
`

`

`U.S. Patent
`
`0a. 14, 1997
`
`Sheet 5 of 6
`
`5,677,171
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`HOSPIRA EX. 1108
`Page 7
`
`

`

`US. Patent
`
`Oct. 14, 1997
`
`Sheet 6 of 6
`
`5,677,171
`
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`HOSPIRA EX. 1108
`Page 8
`
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`
`HOSPIRA EX. 1108
`Page 8
`
`

`

`1
`MONOCLONAL ANTIBODIES DIRECTED
`TO THE HER2 RECEPTOR
`
`5,677,171
`
`This application is a continuation of Ser. No. 07/977,453,
`?led Nov. 18, 1992, now abandoned, which is a continuation
`of Ser. No. 07/147,461, ?led Jan. 25, 1988, now abandoned,
`which is a continuation-in-part of Ser. No. 07/143,912, ?led
`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
`
`This invention is in the ?elds of immunology and cancer
`diagnosis and therapy. More particularly it concerns anti
`bodies speci?cally binding growth factor receptors, hybri
`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
`genesis.
`
`BACKGROUND OF THE INVENTION
`
`15
`
`2
`and c-erbB, are members of the tyrosine-speci?c protein
`kinase family to which many proto-oncogenes belong.
`Recently, a novel transforming gene was identi?ed as a
`result of transfection studies with DNA from chemically
`induced rat neuroblastomas. This gene, called neu, was
`shown to be related to, but distinct from, the c-erbB proto
`oncogene. By means of v-erbB and human EGFR as probes
`to screen human genomic and complementary DNA (cDNA)
`libraries, two other groups independently isolated human
`erbB-related genes that they called HER2 and c-erbB-2
`respectively. Subsequent sequence analysis and chromo
`somal mapping studies revealed that c-erbB-2, and HER2
`are species variants of neu. Afourth group, also using v-erbB
`as a probe, identi?ed the same gene in a mammary carci
`noma cell line, MAC 117, where it was found to be ampli?ed
`?ve- to ten-fold.
`This 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
`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 p11-p13 of chromo
`some 7, where the EGFR gene is located. Also, the HER2
`gene generates a messenger RNA (mRNA) of 4.8 kb, which
`differs from the 5.8- and l0-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
`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
`for an as yet unidenti?ed 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 ampli?cation and in
`some cases rearrangement of the EGF receptor gene. Ampli
`?cation was particularly apparent in squamous carcinomas
`and glioblastomas. The HER2 gene was also found to be
`ampli?ed 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 ampli?ed HER2 gene.
`Although a few sequence rearrangements were detected, in
`most tumors there were no obvious dilferences between
`ampli?ed and normal HER2 genes. Furthermore, ampli?ca
`tion of the HER2 gene correlated signi?cantly with the
`negative prognosis of the disease and the probability of
`relapse.
`To investigate the signi?cance of the correlation between
`overexpression and cellular transformation as it has been
`observed for proto-oncogenes c-mos and N-myc, a HER2
`expression vector and a selection scheme that permitted
`sequence ampli?cation after transfection of mouse NIH 3T3
`cells was employed by Hudziak et al., Proc. Natl. Acad. Sci.
`(USA) 84, 7159 (1987). Ampli?cation of the unaltered
`HER2 gene in NIH 3T3 cells lead to overexpression of p185
`as well as cellular transformation and tumor formation in
`athyrnic mice.
`The effects of antibodies speci?cally binding growth
`factors or growth factor receptors has been studied.
`Examples are discussed below.
`Rosenthal et al., Cell 46, 301 (1986) introduced a human
`TGF-ot cDNA expression vector into established non
`
`25
`
`30
`
`35
`
`40
`
`Macrophages are one of the eifector cell types that play an
`important role in irnmunosurveillance 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
`rophages include reactive oxygen species such as the super
`oxide anion and hydrogen peroxide, arginase, interleukin 1,
`and tumor necrosis factor-0t (TNF-0t). Acquired resistance to
`the toxic elfects 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
`tumorigenesis in vivo and tumor cell growth in vitro. The
`genes encoding TNF-0t and TNF-[3, a structurally related
`cytotoxic protein formerly lmown as lymphotoxin, have
`been cloned and the corresponding proteins expressed in
`Escherichia coil. These proteins display an array of biologi
`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
`phonuclear neutrophil functions, and inhibition of lipid
`biosynthesis. Recently, rHuTNF-ot was shown to augment
`the growth of normal diploid ?broblasts in vitro. The diver
`gent proliferative responses in the presence of rHuTNF-ot
`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-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
`
`45
`
`50
`
`55
`
`65
`
`HOSPIRA EX. 1108
`Page 9
`
`

`

`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
`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 ldnases 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
`speci?cally binding the external domain of the HER2 recep
`tor. The invention also relates to an assay for the HER2
`receptor comprising exposing cells to antibodies speci?cally
`binding the extracellular domain of the HER2 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 elfective amount of antibodies capable of
`inhibiting growth factor receptor function, and a therapeu
`tically eifective 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-ot resistant to TNF-ot, isolating
`those cell which are TNF-ot resistant, screening the isolated
`cells for increased tyrosine kinase activity, and isolating
`receptors and other proteins having increased tyrosine
`kinase activity.
`
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`3
`transformed rat ?broblast cells. Synthesis and secretion of
`TGF-ot by these cells resulted in loss of anchorage
`dependent growth and induced tumor formation in nude
`mice. Anti-human TGF-oc monoclonal antibodies prevented
`the rat cells from forming colonies in soft agar, i.e. loss of
`anchorage dependence. Gill et al. in J. Biol. Chem. 259,
`7755 (1984) disclose monoclonal antibodies speci?c for
`EGF receptor which were inhibitors of EGF binding and
`antagonists of EGF-stimulated tyrosine protein kinase activ
`ity.
`Drebin et al. in Cell 41, 695 (1985) demonstrated that
`exposure of a neu-oncogene-transformed NIH 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) speci?cally binding
`the protein encoded by the neu oncogene signi?cantly
`inhibited the tumorigenic growth of neu-transformed NIH
`3T3 cells implanted into nude mice.
`Akiyama et al. in Science 232, 1644 (1986) raised anti
`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-ot
`and TGF-B synergistically enhance the growth of NRK-49F
`?broblasts, whereas PDGF down regulates EGF receptor
`function on 3T3 cells. Avariety of transformed cells secrete
`factors which are believed to stimulate growth by an auto
`crine mechanism. Sugarrnan et al., Cancer Res. 47, 780
`(1987) demonstrated that under certain conditions, growth
`factors can block the antiproliferative effects of TNF-ot on
`sensitive tumor cells. Speci?cally, epidermal growth factor
`(EGF). and recombinant human transforming growth
`factor-0t (rHuTGF-ot) were shown to interfere with the in
`vitro antiproliferative effects of recombinant human tumor
`necrosis factor-0t (rHuTNF-ot) and -l3 on a human cervical
`carcinoma cell line, ME-l80. The inhibitory effect could be
`observed at EGF or l'HllTGF-Ot. 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
`TNF receptor or to alteration of the a?nity of TNF-ot for its
`receptor. Since the antiproliferative elfect of recombinant
`human interferon-y was not signi?cantly affected by the
`presence of EGF or rHuTGF-ot, the inhibition was speci?c
`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
`cytotoxicity observed when tumor cells were exposed simul
`taneously to rI-IuTNF-ot and recombinant human interferon
`Y. TGF-S can also interfere with the antiproliferative effects
`of rHuTNF-ot in vitro. At concentrations of less than 1
`ng/ml, TGF-B signi?cantly antagonized the cytotoxic effects
`of rHuTNF-ot on NIH 3T3 ?broblasts. Since EGF, platelet
`derived growth factor, and TGF-B all enhanced NIH 3T3 cell
`proliferation, but only TGF-B interfered with rHuTNF-ot
`cytotoxicity, the protective effects of TGF-B were not related
`in a simple manner to enhanced cell proliferation.
`rHuTGF-ot and TGF-B did not have a signi?cant protective
`eifect against rHuTNF-ot-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
`bodies capable of inhibiting growth factor receptor function.
`It is a further object of the invention to provide an
`improved assay for the HER2 receptor.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1a shows TNF-ot resistance of NIH 3T3 cells
`expressing various levels of HER2 p185. FIG. 1b shows
`macrophage cytotoxicity assays for NIH 3T3 cells express
`ing various levels of HER2 p185.
`FIG. 2 demonstrates the level of TNF-ot binding for a
`control cell line (NIH 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 elfect of an
`irrelevant monoclonal antibody (anti-I-IBV) and the e?‘ect of
`monoclonal antibody 4D5 (anti-HER2) on the growth of SK
`BR3 cells in serum.
`FIGS. 5a, 5b and 6a show percent viability of SK B113
`cells as a function of increasing TNF-ot 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. 5a, 5b, 6a and 6b, .--.
`represents TNF-ot alone; H represents antibody (Ab)
`alone; o-o represents 100 Uml TNF-ot and AAb; l:l—l:l
`represents 1000 U/ml TNF-ot and AAb; and A—A represents
`10.000 U/ml TNF-Ot and AAb.
`FIG. 7 shoes percent viability of MDA-MB- l75-VII cells
`as a function of increasing TNF-ot concentration and anti
`
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`5,677,171
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`5
`HER2 p185 monoclonal antibody concentration. In FIG. 7,
`0-. represents TNF-0t alone; I—. represents antibody
`(Ab) alone; o——o represents 100 U/ml TNF-0t and AAb;
`l:|—l:l represents 1000 U/ml TNF-(I and AAb; and A—-A
`represents 10,000 U/ml TNF-0t and AAb.
`FIG. 8 shows percent viability of NIH 3T3 cells overex
`pressing HER2 p185 as a function of increasing TNF-0t
`concentration and anti-HER2 p185 monoclonal antibody
`concentration. In FIG. 8, 0-‘ represents TNF-0L alone;
`I—I represents antibody (Ab) alone; o—o represents 100
`U/ml TNF-0t and AAb; III-El represents 1000 U/ml TNF-0t
`and AAb; and A-A represents 10,000 U/ml TNF-0t and
`AAb.
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`6
`complement and/or mediate antibody-dependent cellu
`lar cytotoxicity (ADCC), e.g. IgG2a antibodies;
`(g) The antibodies which bind the receptor or growth
`factor are conjugated to a toxin (immunotoxins);
`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.
`These antibodies are selected using conventional in vitro
`assays for selecting antibodies which neutralize receptor
`function. Antibodies that act as ligand agonists by mimick
`ing the ligand are discarded by conducting suitable assays as
`will be apparent to those slo'lled in the art. For certain tumor
`cells, the antibodies inhibit an autocrine growth cycle (i.e.
`where a cell secretes a growth factor which then binds to a
`receptor of the same cell). Since some ligands, e.g. TGF-ot,
`are found lodged in cell membranes, the antibodies serving
`a targeting function are directed against the ligand and/or the
`receptor.
`Certain tumor cells secrete growth factors that are
`required for normal cellular growth and division. These
`growth factors, however, can under some conditions stimu
`late unregulated growth of the tinnor cell itself, as well as
`adjacent non-tumor cells, and can cause a tumor to form.
`Epidermal Growth Factor (EGF) has dramatic stimulatory
`effects on cell growth. In puri?ed receptor preparations, the
`EGF receptor is a protein kinase that is activated by the
`binding of EGF. Substrate proteins for this kinase are
`phosphorylated on tyrosine residues. The receptors for
`insulin, platelet-derived growth factor (PDGF) and other
`growth hormones also are tyrosine-speci?c kinases. It is
`believed that ligand binding to the receptor triggers phos
`phorylation of certain proteins by the receptor and in this
`way stimulates cell growth. About one-third of the known
`oncogenes encode proteins that phosphorylate tyrosine resi
`dues on other proteins. It is believed that these oncogene
`products trigger responses analogous to the responses of
`cells to growth factors and hormones. The erbB oncogene
`product is a portion of the EGF receptor that lacks the
`hormone-binding domain and may give rise to a constitutive
`growth-stimulating signal.
`One embodiment of this invention is a method of inhib
`iting the growth of tumor cells by administering to a patient
`a therapeutically effective amount of antibodies that inhibit
`the HER2 receptor biological function of tumor cells.
`Overexpression of growth factor receptors increases the
`resistance of cells to TNF as demonstrated below._ Overex
`pres sion of the HERl receptor (EGF receptor), met receptor
`like protooncogene product, and HER2 receptor all show
`this increased resistance. It is shown in the Examples below
`that ampli?ed expression -of HER2, which encodes the
`HER2 receptor (p185), induces resistance of NIH 3T3 cells
`to the cytotoxic effects of macrophages or TNF-(1. Induction
`of NIH 3T3 cell resistance to TNF-0t by overexpression of
`p185 is accompanied by alterations in the binding of TNF-0t
`to its receptor. Overexpression of p185 is also associated
`with resistance of certain human breast tumor cell lines to
`the cytotoxic effects of TNF-oi.
`In another embodiment of the invention, tumor cells are
`treated by (l) administering to a patient antibodies directed
`against the growth factor and/or its receptor, that inhibit the
`biological function of the receptor and that sensitize the cells
`to cytotoxic factors such as TNF, and (2) administering to
`the patient cytotoxic factor(s) or other biological response
`modi?ers which activate immune system cells directly or
`indirectly to produce cytotoxic factors.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`A new application of antibodies to inhibit the growth of
`tumor cells has been discovered. Surprisingly it has been
`found that by inhibiting growth factor receptor function, e. g.
`the HER2 receptor function, cell growth is inhibited, and the,
`cells are rendered more susceptible to cytotoxic factors.
`Thus, for example, breast cancer cells which are refractory
`to TNF-oi alone can be made susceptible to TNF-oi if the
`cells are ?rst treated with antibodies which inhibit growth
`factor receptor f