PHIGENIX
`Exhibit 1017
`
`

`
`United States Patent
`
`[19]
`
`[11] Patent Number:
`
`5,770,195
`
`Hudziak et al.
`
`[45] Date of Patent:
`
`*Jun. 23, 1998
`
`US005770195A
`
`[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.
`
`[ * ] Notice:
`
`The term of this patent shall not extend
`beyond the expiration date of Pat. No.
`5,725,856, and 5,720,954.
`
`[21] Appl. No.: 447,517
`
`[22]
`
`Filed:
`
`May 23, 1995
`
`Related U.S. Application Data
`
`[63] Continuation of Ser. No. 286,303, Aug. 5, 1994, Pat. No.
`5,677,171, which is a continuation of Ser. No. 977,453, Nov.
`18, 1992, abandoned, 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 ...................... .. A61K 39/395; C07K 16/30;
`C07K 16/46; C12P 21/08
`................................... .. 424/130.1; 424/133.1;
`[52] U.S. Cl.
`424/134.1, 424/142.1, 424/143.1, 424/155.1,
`424/156.1; 424/174.1; 424/183.1; 530/388.22;
`530/387.3; 530/387.7; 530/388.8
`[58] Field of Search ............................ .. 424/130.1, 183.1,
`424/174.1, 156.1, 155.1, 143.1, 142.1, 134.1,
`133.1; 530/388.22, 387.3, 387.7, 388.8
`
`[56]
`
`References Cited
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`.
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`.
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`3/1990 Honn et al.
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`.
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`.
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`11/1995 Herlyn et al.
`.
`
`.
`
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`
`(List continued on next page.)
`
`Primary Examiner—Lila Feisee
`Assistant Examiner—Geetha P. Bansal
`
`Attorney, Agent, or Firm—Wendy M. Lee
`
`[57]
`
`ABSTRACT
`
`A method of inhibiting growth of tumor cells which over-
`express a growth factor receptor or growth factor by treat-
`ment of the cells with antibodies which inhibit the growth
`factor receptor function, is disclosed. A method of treating
`tumor cells with antibodies which inhibit growth factor
`receptor function, and with cytotoxic factor(s) such as tumor
`necrosis factor, is also disclosed. By inhibiting growth factor
`receptor functions tumor cells are rendered more susceptible
`to cytotoxic factors.
`
`36 Claims, 6 Drawing Sheets
`
`PHIGENIX
`
`Exhibit 1017-01
`
`359282 A2
`239400
`667165 A1
`60—243027
`62—108157
`W0 85/03357
`W0 87/07646
`W0 89/10412
`
`...... .. C12N 15/00
`
`.
`
`.
`
`3/1990 European Pat. Off.
`8/1994 European Pat. Off.
`8/1995 European Pat. Off.
`12/1985
`Japan .
`5/1987
`Japan .
`8/1985 WIPO ........................ .. G01N 33/574
`12/1987 WIPO .
`11/1989 WIPO ............................ .. C12Q 1/68
`
`OTHER PUBLICATIONS
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`
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`Akiyama et al., “The product of the human c—erbB—2 Gene:
`a 185—Kilodalton Glycoprotein with tyrosine Kinase Activ-
`ity” Science 232:1644—1646 (1986).
`
`

`
`5,770,195
`Page 2
`
`OTHER PUBLICATIONS
`
`Kipps et al., “Schemata for the production of monoclonal
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`Mansi et al., “In vivo Evaluation of an Anti-Melanoma
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`Anti-Epidermal Growth Factor Receptor Monoclonal Anti-
`bodies with Different
`Isotypes” Cancer Research
`46:5592-5598 (1986).
`McKenzie et al., “Generation and characterization of mono-
`clonal antibodies specific for the human neu oncogene
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`Morrison, “Transfectomas Provide Novel Chimeric Anti-
`bodies” Science 229:1202-1207 (1985).
`Muller et al., “Single-Step Induction of Mammary Adeno-
`carcinoma in Transgenic Mice Bearing the Activated c—neu
`Oncogene” Cell 542105-115 (1988).
`Myers et al., “Biological Effects of Monoclonal Antireceptor
`Antibodies Reactive with neu Oncogene Product, p185”e“”
`Methods in Enzymology 198:277-290 (1991).
`Padhy et al., “Identification of a Phosphoprotein Specifically
`Induced by the Transforming DNA of Rat Neuroblastomas”
`Cell 282865-871 (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-
`nologically Mediated
`and Effector Cell-independent
`Effects” Cancer Research 47(14) 23692-3696 (1987).
`Rosenthal et al., “Expression in Rat Fibroblasts of a Human
`Transforming Growth Factor-ot 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. 12511-529 (1983).
`Schechter et al., “The neu Gene: An erbB-Homologous
`Gene Distinct from and Unlinked to the Gene Encoding the
`EGF Receptor” Science 2292976-978 (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-1/epidermal growth factor-
`receptor gene and is amplified in a human salivary gland
`adenocarcinoma” Proc. Natl. Acad. Sci. USA 8226497-6501
`
`(1985).
`Slamon et al., “Human Breast Cancer: Correlation of
`Relapse and Survival with Amplification of the HER-2/neu
`Oncogene” Science 2352177-182 (1987).
`Sobol et al., “Epidermal Growth Factor Receptor Expression
`in Human Lung Carcinomas Defined by a Monoclonal
`Antibody” J. Natl. Cancer Institute 79(3):403-407 (1987).
`Sugarman et al., “Effects of Growth Factors on the Antipro-
`liferative Activity of Tumor Necrosis Factor” Cancer
`Research 472780-786 (1987).
`
`Sugarman et al., “Recombinant Human Tumor Necrosis
`Factor-ot: Effects on Proliferation of Normal and Trans-
`
`formed Cells in Vitro” Science 2302943-945 (1985).
`Takahashi et al., “Radioimmunodetection of Human Glioma
`Xenografts by Monoclonal Antibody to Epidermal Growth
`Factor Receptor” Cancer Reserch 4723847-3850 (1987).
`Urban et al., “Tumor necrosis factor: A potent effector
`molecule for tumor cell killing by activated macrophages”
`Proc. Natl. Acad. Sci. 8325233-5237 (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 Biolog
`7(5):2019-2023 (1987).
`Venter et al., “Overexpression of the c-erbB-2 Oncoprotein
`in Human Breast Carcinomas: Immunohistological Assess-
`ment Correlates with Gene Amplification” Lancet pps.
`69-72 (1987).
`Vitetta et al., “Redesigning nature’s poisons to create anti-
`tumor reagents” Science 23821098-1104 (1987).
`Vollmar et al., “Toxicity of Ligand and Antibody-Directed
`Ricin A-Chain Conjugates Recognizing the Epidermal
`Growth
`Factor Receptor”
`J. Cellular Physiolog
`1312418-425 (1987).
`Yamamoto et al., “Similarity of protein encoded by the
`human c-erb-B-2 gene to epidermal growth factor recep-
`tor” Nature 3192230-34 (1986).
`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-
`therapeutic Agenst” J. Natl. Canc. Inst 63(5):1185-1188
`(1979).
`Bernards et al., “Effective Tumor Immunotherapy Directed
`Against an Oncogene-encoded Product Using a Vaccinia
`Virus Vector” Proc. Natl. Acad. Sci. USA 8426854-6858
`
`(Oct. 1987).
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`Oncol. Nurs. (abstract only) 3(1):67-73 (1987).
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`domains of the neu oncogene-encoded p185 molecule exert
`syncrgistic anti—tumor effects in vivo” Oncogene 22273-277
`(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 5124575-4580 (Sep. 1,
`1991).
`Masuko et al., “A murine Monoclonal Antibody That Rec-
`ognizes an Extracellular Domain of the Human c-erbB-2
`Protooncogene Product” Jpn J. Cancer Res. 80:10-14 (Jan.
`1989).
`Park et al., “Development of anti-p185HER2 Immunolipo-
`somes for Cancer Therapy” Proc. Natl. Acad. Sci. USA
`9221327-1331 (Feb. 1995).
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`Structure, Expression and Homology to Lymphotoxin”
`Nature 3122724-729 (1984).
`Ring et al., “Identity of BCA200 and c-erbB-2 Indicated by
`Reactivity of Monoclonal Antibodies with Recombinant
`c-erbB-2” Molecular Immunology 28(8):915-917 (1991).
`
`PHIGENIX
`
`Exhibit 1017-02
`
`

`
`5,770,195
`Page 3
`
`Arteaga et al., “Antibodies Against pl85/HER2 Enhance
`Etoposide—Induced Cytotoxicity Against Human Breast
`Carcinoma Cells” Proceedings ofASCO—29th Annual Meet-
`ing (Abstract #101), Orlando, FL 12:75 (1993).
`Bargmann et al., “Multiple Independent Activations of the
`neu Oncogene by a Point Mutation Altering the Transmem-
`brane Domain of p185” Cell 45:649—659 (Jun. 6, 1986).
`Baselga et al., “Anti Her2 Humanized Monoclonal Antibody
`(MAb) Alone and in Combination with Chemotherapy
`Against Human Breast Carcinoma Xenografts” Proceedings
`ofASCO—Z3th Annual Meeting (Abstract #53), Dallas, TX
`13:63 (1994).
`Baselga et al., “Phase II Study of Weekly Intravenous
`Recombinant Humanized Anti—p185/HER2 Monoclonal
`Antibody in Patients With HER2/neu—OVereXpressing
`Metastatic Breast Cancer” J. Clin. Oncol. 14(3):737—744
`(1996).
`Baselga et al., “Receptor Blockade With Monoclonal Anti-
`bodies
`as Anti—Cancer
`Therapy” Pharmac.
`Ther.
`64:127—154 (1994).
`the Epidermal
`Beguinot et al., “Down—Regulation of
`Growth Factor Receptor in KB Cells is Due to Receptor
`Internalization and Subsequent Degradation in Lysozymes”
`Proc. Natl. Acad. Sci. USA 81(8):2384—2388 (1984).
`De Santes et al., “Radiolabeled Antibody Targeting of the
`HER—2/neu
`Oncoprotein—l”
`Cancer
`Research
`52:1916—1923 (1992).
`
`Lyall et al., “EGF Induces Receptor Down—regulation with
`No Receptor Recycling in KB Cells” J. Cell Physiol
`122(l):166—170 (1985).
`
`Pegram et al., “Monoclonal Antibody to HER—2/neu Gene
`Product Potentiates Cytotoxicity of Carboplatin and DoXo-
`rubicin in Human Breast Tumor Cells” Proceedings of the
`American Assoc.
`for Cancer Research (Abstract #
`2639—83rd Annual Meeting), San Diego, CA 33:442 (1992).
`
`Pegram et al., “Phase II Study of Intravenous Recombinant
`Humanized Anti—p185 Monoclonal” Proceedings of the
`ASCO—3Z st Annual Meeting (Abstract #124), Los Angeles,
`CA 14:106 (1995).
`
`Pietras et al., “Antibody to HER—2/neu Receptor Blocks
`DNA Repair After Cisplatin in Human Breast and Ovarian
`Cancer Cells” Oncogene 9:1829—1838 (1994).
`
`Shepard et al., “Monoclonal Antibody Therapy of Human
`Cancer: Taking the HER2 Protooncogene to the Clinic” J.
`Clin. Immunol. 11(3):117—127 (1991).
`
`Taetle et al., “Effects of Anti—Epidermal Growth Factor
`(EGF) Receptor Antibodies and an Anti EGF Receptor
`Rocombinant—ricin A Chain Immunoconjugate on Growth
`of
`Human
`Cells”
`J.
`Natl.
`Cancer
`Institute
`
`80(13):1053—1059 (1988).
`
`PHIGENIX
`
`Exhibit 1017-03
`
`

`
`U.S. Patent
`
`Jun. 23, 1998
`
`Sheet 1 of 6
`
`5,770,195
`
`0..
`
`5‘
`
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`
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`
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`
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`11:1
`EFFECTOR:TARGET CELL RATIO
`
`PHIGENIX
`
`Exhibit 1017-04
`
`

`
`U.S. Patent
`
`Jun. 23, 1998
`
`Sheet 2 of 6
`
`5,770,195
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`PHIGENIX
`
`Exhibit 1017-05
`
`

`
`U.S. Patent
`
`Jun. 23, 1998
`
`Sheet 3 of 6
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`PHIGENIX
`
`Exhibit 1017-06
`
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`
`U.S. Patent
`
`Jun. 23, 1998
`
`Sheet 4 of 6
`
`5,770,195
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`PHIGENIX
`
`Exhibit 1017-07
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`
`U.S. Patent
`
`Jun. 23, 1998
`
`Sheet 5 of 6
`
`5,770,195
`
`Fig.6u
`
`
`
`RELATIVEPERCENTVIABILITY(RPV)
`
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`
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`
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`
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`
`PHIGENIX
`
`Exhibit 1017-08
`
`

`
`U.S. Patent
`
`Jun. 23, 1998
`
`Sheet 6 of 6
`
`5,770,195
`
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`
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`
`PHIGENIX
`
`Exhibit 1017-09
`
`

`
`5,770,195
`
`1
`MONOCLONAL ANTIBODIES DIRECTED
`TO THE HER2 RECEPTOR
`
`CROSS REFERENCES
`
`This application is a continuation of U.S. application Ser.
`No. 08/286,303 filed 5 Aug. 1994(U.S. Pat. No. 5,677,877),
`which application is a continuation of U.S. application Ser.
`No. 07/977,453 filed 18 Nov. 1992 (abandoned), which
`application is a continuation of U.S. application Ser. No.
`07/147,461 filed 25 Jan. 1988 (abandoned), which applica-
`tion is a continuation-in-part of U.S. application Ser. No.
`07/143,912 filed 12 Jan. 1988 (abandoned), which applica-
`tions are incorporated herein by reference and to which
`applications priority is claimed under 35 USC §120.
`
`FIELD OF TIIE 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 kinases 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-ot (TNF-01). 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-01 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-(X and TNF-[3, a structurally related
`cytotoxic protein formerly known as lymphotoxin, have
`been cloned and the corresponding proteins expressed in
`Escherichia coli. 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, IHUTNF-(X was shown to augment
`the growth of normal diploid fibroblasts in vitro. The diver-
`gent proliferative responses in the presence of IHUTNF-(X
`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
`B chain of platelet-derived growth factor (PDGF); c-fms,
`which is homologous to the transforming gene of the feline
`
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`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 avian erythroblastosis virus
`(v-erbB). The two receptor-related proto-oncogenes, c-fms
`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.
`
`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 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 differences 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.
`
`PHIGENIX
`
`Exhibit 1017-10
`
`

`
`5,770,195
`
`3
`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-01 cDNA expression vector into established non-
`transformed rat fibroblast cells. Synthesis and secretion of
`TGF-01 by these cells resulted in loss of anchorage-
`dependent growth and induced tumor formation in nude
`mice. Anti-human TGF-(X 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-tranformed 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 treat-
`ment with a monoclonal antibody (IgG2a isotype) specifi-
`cally binding the protein encoded by the neu oncogene
`significantly 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-(X
`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-01 on
`sensitive tumor cells. Specifically, epidermal growth factor
`(EGF) and recombinant human transforming growth
`factor—ot (rHuTGF—(x) were shown to interfere with the in
`vitro antiproliferative effects of recombinant human tumor
`necrosis factor-ot (rHuTNF-01) and -[3 on a human cervical
`carcinoma cell line, ME-180. The inhibitory effect could be
`observed at EGF or IHUTGF-(X 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 afiinity of TNF-01 for its
`receptor. Since the antiproliferative effect of recombinant
`human interferon-y was not significantly affected by the
`presence of EGF or FHUTGF-(X, 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 IHUTNF-(X and recombinant human interferon-
`y. TGF-[3 can also interfere with the antiproliferative effects
`of IHUTNF-(X in vitro. At concentrations of less than 1
`
`ng/ml, TGF-[3 significantly antagonized the cytotoxic effects
`of IHUTNF-(X on NIH 3T3 fibroblasts. Since EGF, platelet-
`derived growth factor, and TGF-[3 all enhanced NIH 3T3 cell
`proliferation, but only TGF-[3 interfered with IHUTNF-(X
`cytotoxicity, the protective effects of TGF-[3 were not related
`in a simple manner
`to enhanced cell proliferation.
`IHUTGF-(X and TGF-[3 did not have a significant protective
`effect against rHuTNF-01-mediated cytotoxicity on two other
`tumor cell lines, BT-20 and L-929 cells.
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`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 thc HER2 rcccptor.
`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.
`
`Astill further object of the invention is to provide an assay
`for tyrosine kinases that may have a role in tumorigenesis.
`Other objects, features and characterisitics 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-
`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 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-(X resistant to TNF-01, isolating
`those cell which are TNF-(X 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. 1a shows TNF-(X 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-(X binding for a
`control cell line (NIH 3T3 neo/dhfr) and for a cell line
`overexpressing HER2 p185 (HER2—38OO).
`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. 5a, 5b and 6a show percent viability of SK BR3
`cells as a function of increasing TNF-01 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.
`FIG. 7 shoes percent viability of MDA-MB-175-VII cells
`as a function of increasing TNF-01 concentration and anti-
`HER2 p185 monoclonal antibody concentration.
`
`PHIGENIX
`
`Exhibit 1017-11
`
`

`
`5,770,195
`
`5
`FIG. 8 shows percent viability of NIH 3T3 cells overex-
`pressing HER2 p185 as a function of increasing TNF-(X
`concentration and anti-HER2 p185 monoclonal antibody
`concentration.
`
`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-(X alone can be made susceptible to TNF-01 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-01.
`
`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. Abnormal 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
`effects of a cytotoxic factor such as TNF-01;
`(e) The antibodies inhibit the tyrosine kina