United States Patent
`Hudziak et al.
`
`[191 4
`
`[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 U.S. 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.
`
`Int. Cl.5 ......................... C07K 16/00; G01N 33/574
`[51]
`[52] U.S. Cl. .............................. 435/240.27; 530/388.8;
`
`530/388.85; 530/387.7; 435/7.23; 435/172.2
`[58] Field of Search .............................. 530/387.7, 388.8,
`530/388.85, 381.1; 435/723, 240.27, 172.2,
`70.21
`
`[56]
`
`References Cited
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`US005677171A
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`[11] Patent Number:
`
`5,677,171
`
`[45] Date of Patent:
`
`Oct. 14, 1997
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`Hudziak et al., “p185HER2 Monoclonal Antibody Has Anti-
`proljferative Effects In Vitro and Sensitizes Human Breast
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`King et a1. , “Amplification of a Novel v—erbB—Related Gene
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`(1985).
`Kipps et al., “Schemata for the production of monoclonal
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`tal Immunology, Herzenberg et al. (eds), Blackwell Sci.,
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`(1985).
`
`(List continued on next page.)
`
`Primary Examiner—L1'la Feisee
`Assistant Examiner—Geetha P. Bansal
`Artomey, Agent, or Firm—Wendy M. Lee
`[57]
`ABSTRACT
`
`Monoclonal antibodies which bind to the extracellular
`domain of the I-IER2 receptor and inhibit growth of
`SK-BR-3 breast tumor cells, which overexpress I-lER2, are
`disclosed. The monoclonal antibodies can be used for in
`vino assays for detecting a tumor characterized by amplified
`expression of I~lER2.
`
`39 Claims, 6 Drawing Sheets
`
`PETITIONER'S EXHIBITS
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1096 Page 1 of 19
`Exhibit 1096 Page 1 of 19
`
`

`
`5,677,171
`Page 2
`
`OTHER PUBLICATIONS
`
`Masui et al., “Mechanism of Antitumor Activity in Mice for
`Anti-Epidermal Growth Factor Receptor Monoclonal Anti-
`bodies with Dilferent
`Isotypes” Cancer Research
`465592-5598 (1986).
`Morrison, ‘Transfectomas Provide Novel Chimeric Anti-
`bodies” Science 2129:1202-1207 (1985).
`Padhy et al., “Identification of a Phosphoprotein Specifically
`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 Biachem. 35(4):3l5-320 (1987).
`Rodeck et al., “Tumor Growth Modulation by a Monoclonal
`Antibody to the Epidermal Growth Factor Receptor: Irnmu-
`nologically Mediated and Eifector Cell-independent
`Effects” Cancer Research 47(14):3692-3696 (1987).
`Rosenthal et al., “Expression in Rat Fibroblasts of a Human
`Transforming Growth Factor-or cDNA Results in Transfor-
`mation” Cell 462301-309 (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—Homo1ogous
`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 463225-3238 (1986).
`Semba et al., “A v-erbB-related protooncogene c-erbB—2,
`is distinct from the c-erb-B-1/epidermal growth factor-
`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:l77-182 (1987).
`Sobol et al. , “Epidermal Growth Factor Receptor Expression
`in Human Lung Carcinoma Defined by a Monclonal Anti-
`body” J. Natl. Cancer Institute 79(3):403—407 (1987).
`Sugarman et al., “Efl’ects of Growth Factors on the Antipro-
`liferative Activity of Tumor Necrosis Factor” Cancer
`Research 471780-786 (1987).
`Sugarman et al., “Recombinant Human Tumor Necrosis
`Factor—0t: Effects on Proliferation of Normal and Trans-
`fonned 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., “Amplification of the neu (c—erbB—2)
`Oncogene in Human Mammary Tumors Is Relatively Fre-
`quent and Is Often Accompanied by Amplification 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 Amplification” Lancet pp. 69-72
`(1987).
`
`Vitetta et al., “Redesigning natnre’s poisons to create anti-
`tumor reagents” Science 238:l098—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 specific for the human neu oncogene
`product, p185” Oncogene 4:543-548 (1989).
`Muller et al., “Single-Step Induction of Mammary Adeno-
`carcinoma in Transgenic Mice Bearing the Activated c—neu
`Oncogene” Cell 54:l05-115 (1988).
`Myers et al., “Biological Effects of Monoclonal Antireceptor
`Antibodies Reactive with neu Oncogene Product, pl85"""’
`Methods in Enzymology 198 :277-290.
`Drebin et aL, “Monoclonal Antibodies Specific for the neu
`Oncogene Product Directly Mediate Anti-tumor Efiects In
`Vivo” Oncogene 2 (4):387-394 (1988).
`Hudziak et al., “Amplified Expression of the HER2/ERBB2
`Oncogene Induces Resistance to Tumor Necrosis Factor 0: 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—l6ll (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):1l85-1188
`(1979).
`‘
`Bernards et al., “Effective Tumor Immunotherapy Directed
`Against an Oncogene—encoded Product Using a Vaccine
`Virus Vector” Proc. Natl. Acad. Sci. USA 84:6854—6358
`(Oct. 1987).
`Bucholtz, J.D., “Radiolabe1ed Antibody Therapy” Semin.
`Oneal. 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 efiects in vivo” Oncogene 2:273-277
`(1988).
`the
`Hancock et al., “A Monoclonal Antibody Against
`c-erbB—2 Protein Enhances the Cytotoxicity of cis-Diam-
`minedichloroplatinum Against Human Breast and Ovarian
`Tumor Cell Lines” Cancer Research 5l:4575—4580 (Sep. 1,
`1991).
`Masuko et al., “A murine Monoclonal Antibody ‘That Rec-
`ognizes an Extracellular Domain of the Human c—erB-2
`Protooncogene Product” Jpn J. Cancer Res. 80:10-14 (Jan.
`1989).
`Pennica et al., “Human Tumour Necrosis Factor: Precursor
`Structure, Expression and Homology to Lymphotoxin”
`Nature 312:724—729 (1984).
`Ring et al., “Identity of BCAZOO and c—erB-2 Indicated by
`Reactivity of Monoclonal Antibodies with Recombinant
`c-erbB—2” Molecular Immunology 28(8):915-917 (1991).
`King, C.R. DHHS Patent Application 6/836.414. National
`Technical Information Service, Springfield, VA. 22161 Pub."
`86—197928.
`
`PETITIONER'S EXHIBITS
`PETITIONER'S EXHIBITS
`
`Exhibit 1096 Page 2 of 19
`Exhibit 1096 Page 2 of 19
`
`

`
`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 1 of 6
`
`5,677,171
`
`Fca
`
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`
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`
`PETITIONER'S EXHIBITS
`PETITIONER'S EXHIBITS
`
`Exhibit 1096 Page 3 of 19
`Exhibit 1096 Page 3 Of 19
`
`

`
`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 2 of 6
`
`5,677,171
`
`Fig.3INHIBITIONOFSKBR3GROWTHBYANTI-HER-2MABS
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`PETITIONER'S EXHIBITS
`PETITIONER'S EXHIBITS
`
`Exhibit 1096 Page 4 of 19
`Exhibit 1096 Page 4 Of 19
`
`

`
`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 3 of 5
`
`5,677,171
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`PETITIONER'S EXHIBITS
`PETITIONER'S EXHIBITS
`
`Exhibit 1096 Page 5 of 19
`Exhibit 1096 Page 5 of 19
`
`

`
`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 4 of 6
`
`5,677,171
`
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`PETITIONER'S EXHIBITS
`PETITIONER'S EXHIBITS
`
`Exhibit 1096 Page 6 of 19
`Exhibit 1096 Page 6 Of 19
`
`

`
`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 5 of 6
`
`5,677,171
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`PETITIONER'S EXHIBITS
`PETITIONER'S EXHIBITS
`
`Exhibit 1096 Page 7 of 19
`Exhibit 1096 Page 7 of 19
`
`

`
`U.S. Patent
`
`Oct. 14, 1997
`
`Sheet 6 of 6
`
`5,677,171
`
`Fig.7
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`PETITIONER'S EXHIBITS
`PETITIONER'S EXHIBITS
`
`Exhibit 1096 Page 8 of 19
`Exhibit 1096 Page 8 of 19
`
`

`
`1
`MONOCLONAL ANTIBODIES DIRECTED
`TO THE HER2 RECEPTOR
`
`This 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.
`
`10
`
`FIELD OF THE INVENTION
`
`This invention is in the fields of immunology and cancer
`diagnosis and therapy. More particularly it concerns anti-
`bodies specifically 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 lcinases that are involved in tumori-
`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-
`rophages include reactive oxygen species such as the super-
`oxide anion and hydrogen peroxide, arginase. interleukin 1,
`and tumor necrosis factor-or (TNF-ct). 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
`tumorigenesis in vivo and tumor cell growth in Vit1'o. The
`genes encoding TNF-or 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- , activation of human polymor-
`phonuclear neutrophil functions, and inhibition of lipid
`biosynthesis. Recently, rHuTNF-cc was shown to augment
`the growth of normal diploid fibroblasts in vitro. The diver-
`gent proliferative responses in the presence of rHuTNF—oL
`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 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
`
`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
`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, identified the same gene in a mammary carci-
`noma cell line, MAC 117, where it was found to be amplified
`five- to ten-fold.
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`This gene, which wfll 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 q2l 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 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
`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 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-
`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 dilferences between
`amplified and normal HER2 genes. Furthermore, amplifica-
`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
`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 NIH 3T3
`cells was employed by Hudziak et al., Proc. Natl. Acad. Sci.
`(USA) 84, 7159 (1987). Amplification of the unaltered
`HER2 gene in NIH 3T3 cells lead to overexpression 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.
`Examples are discussed below.
`.
`Rosenthal et al., Cell 46. 301 (1986) introduced a human
`TGF-ot CDNA expression vector into established non-
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`transfonned rat fibroblast cells. Synthesis and secretion of
`TGF—0t by these cells resulted in loss of anchorage-
`dependent growth and induced tumor formation in nude
`mice. Anti-human TGF-cc 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 specific 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) specifically binding
`the protein encoded by the neu oncogene significantly
`inhibited the tumorigenic growth of neu-transformed NIH
`3T3 cells implanted into nude mice.
`Akiyarna 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—0t
`and TGF-[3 synergistically enhance the growth of NRK-49F
`fibroblasts, 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 Sugarman et al., Cancer Res. 47, 780
`(1987) demonstrated that under certain conditions, growth
`factors can block the antiproliferative effects of TNF-0t on
`sensitive tumor cells. Specifically, epidermal growth factor
`(EGF). and recombinant human transforming growth
`factor-ot (IHHTGF-Ot) were shown to interfere with the in
`vitro antiproliferative effects of recombinant human tumor
`necrosis factor-or (rHuTNF-ot) and -[3 on a human cervical
`carcinoma cell line, ME—l80. The inhibitory eifect could be
`observed at EGF or rHuTGF-or 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 aflinity of TNF-ot for its
`receptor. Since the antiproliferative eifect of recombinant
`human interferon-y was not significantly affected by the
`presence of EGF or rHuTGF-ot, 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
`cytotoxicity observed when tumor cells were exposed simul-
`taneously to rHuTNF—oL and recombinant human interferon-
`Y. TGF-6 can also interfere with the antiproliferative effects
`of rHuTN'F-ot in vitro. At concentrations of less than 1
`ng/ml, TGF-[3 significantly antagonized the cytotoxic eifects
`of rHuTNF-ot on NIH 3T3 fibroblasts. Since EGF, platelet-
`derived growth factor, and TGF-B all enhanced NIH 3T3 cell
`proliferation, but only TGF—l3 interfered with rHuTNF—0L
`cytotoxicity, the protective etfects of TGF-[3 were not related
`in a simple manner to enhanced cell proliferation.
`rHuTGF-ot and TGF—B did not have a significant protective
`etfect against rHuTNF-ct-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.
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`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.
`SUNEMARY OF THE INVENTION
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`The subject invention relates to monoclonal antibodies
`specifically 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 specifically
`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 effective amount of antibodies capable of
`inhibiting growth factor receptor function, and a therapeu-
`tically efiective 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-ct resistant to TNF—0., isolating
`those cell which are TNF—oc resistant, screening the isolated
`cells for increased tyrosine lcinase activity, and isolating
`receptors and other proteins having increased tyrosine
`kinase activity.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1a shows TNF-on 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—oL 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 effect of an
`irrelevant monoclonal antibody (anti-I-IBV) and the eifect 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 BR3
`cells as a function of increasing TNF-on 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, O——O
`represents TNF-on alone; I——I represents antibody (Ab)
`alone; o—o represents 100 Uml TNF-cc and AAb; I
`represents 1000 U/ml TNF-oz and AAb; and A—A represents
`10,000 U/ml TNF-on and AAb.
`FIG. 7 shoes percent viability of MDA—MB-175-VII cells
`as a function of increasing TNF-or concentration and anti-
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`HER2 p185 monoclonal antibody concentration. In FIG. 7,
`I—O represents TN'F—ot alone; I—I represents antibody
`(Ab) alone; o—o represents 100 U/ml TNF-on and AAb;
`E|—E represents 1000 U/ml TNF-or and AAb; and A—A
`represents 10,000 U/ml TNF-Oi and AAb.
`FIG. 8 shows percent viability of NIH 3T3 cells overex-
`pressing I-IER2 p185 as a function of increasing TNF—0t
`concentration and anti-HER2 p185 monoclonal antibody
`concentration. In FIG. 8, O——O represents TNF-ot alone;
`I—I represents antibody (Ab) alone; o—c represents 100
`U/ml TNF-on and AAb; El—E| represents 1000 Ulml TNF-ot
`and AAb; and A—A represents 10,000 U/ml TNF-oz and
`AAb.
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`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—oL alone can be made susceptible to TNF-(1 if the
`cells are first treated with antibodies which inhibit growth
`factor receptor function. The increase of susceptibility has
`been demonstrated using the HER2 receptor and monoclonal
`antibodies directed against the HER2 receptor, and tumor
`necrosis factor-ot.
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`’ The method of this invention is useful in the therapy of
`malignant or benign tumors of mammals where the abnor-
`mal growth rate of the tumor is dependent upon growth
`factor receptors. Abnonnal growth rate is a rate of growth
`which is in excess of that required for normal homeostasis
`and is in excess of that for normal tissues of the same origin.
`Many of these tumors are dependent upon extracellular
`sources of the growth factor recognized by the receptor, or
`upon synthesis of the growth factor by the tumor cell itself.
`This latter phenomenon is termed “autocrine” growth.
`The methods of the subject invention is applicable where
`the following conditions are met:
`(1) the growth factor receptor and./or ligand (growth
`factor) is expressed, and tumor cell growth depends
`upon the growth factor receptor biological function;
`(2) antibodies specifically binding the growth factor
`receptor and/or ligand inhibit the growth factor receptor
`biological function.
`While not wishing to be constrained to any particular
`theory of operation of the invention, it is believed that the
`antibodies inhibit growth factor receptor biological function
`in one or more of the following ways:
`(a) The antibodies bind to the extracellular domain of the
`receptor and inhibit the ligand from binding the recep-
`tor;
`
`(b) The antibodies bind the ligand (the growth factor)
`itself and inhibit the ligand from binding the receptor;
`(c) The antibodies down regulate the growth factor recep-
`tor;
`
`(d) The antibodies sensitize tumor cells to the cytotoxic
`efiects of a cytotoxic factor such as TNF-ot;
`(e) The antibodies inhibit the tyrosine kinase activity of
`the receptor.
`In cases (f) and (g), the antibodies inhibit growth factor
`receptor biological function indirectly by mediating cyto-
`toxicity via a targeting function:
`(f) The antibodies belong to a sub-class or isotype that
`upon complexing with the receptor activates serum
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`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 sldlled 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