Case 1:18-cv-00924-CFC Document 312-1 Filed 07/19/19 Page 1 of 287 PageID #: 23798
`Case 1:18—cv-00924-CFC Document 312-1 Filed 07/19/19 Page 1 of 287 PageID #: 23798
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`EXHIBIT 1
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`EXHIBIT 1
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`I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111
`Case 1:18-cv-00924-CFC Document 312-1 Filed 07/19/19 Page 2 of 287 PageID #: 23799
`US006627196Bl
`
`(12) United States Patent
`Baughman et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,627,196 Bl
`Sep.30,2003
`
`(54) DOSAGES FOR TREATMENT WITH ANTI(cid:173)
`ERBB2 ANTIBODIES
`
`(75)
`
`Inventors: Sharon A. Baughman, Ventura, CA
`(US); Steven Shak, Burlingame, CA
`(US)
`
`(73) Assignee: Genentech, Inc., South San Francisco,
`CA (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`(21) Appl. No.: 09/648,067
`
`(22) Filed:
`
`Aug. 25, 2000
`
`Related U.S. Application Data
`( 60) Provisional application No. 60/213,822, filed on Jun. 23,
`2000, and provisional application No. 60/151,018, filed on
`Aug. 27, 1999.
`
`(51) Int. Cl.7 .............................................. A61K 39/395
`(52) U.S. Cl. .......................... ... ... 424/138.1; 424/ 131.1;
`424/ 133.1; 424/134.1; 424/135.1; 424/ 136.1;
`424/ 137.1; 424/139.1; 424/141.1; 424/ 142.1;
`424/ 143.1; 424/144.1; 424/145.1; 424/ 146.1;
`424/ 147.1; 424/150.1; 424/151.1; 424/ 152.1;
`424/ 153.1; 424/154.1; 424/155.1; 424/ 156.1;
`424/ 158.1; 424/172.1; 424/174.1
`(58) Field of Search ........................... 424/ 130.1, 138.1,
`424/ 141.1, 142.1, 152.1, 155.1, 131.1, 133.1,
`134.1, 135.1, 136.1, 137.1, 139.1, 143.1,
`144.1, 145.1, 146.1, 147.1, 150.1, 151.1,
`153.1, 154.1, 156.1, 158.1, 172.1, 174.1
`
`(56)
`
`References Cited
`
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`6,333,348 Bl * 12/2001 Vogel et al.
`................ 514/449
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`EP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`
`0003089 Al
`0599274 Al
`616812 Al
`0711565 Bl
`3-240498
`5-117165
`5-170667
`5-213775
`5-317084
`95006982 B2
`7-59588
`2761543 B2
`2895105 B2
`
`7/1979
`6/1994
`9/1994
`8/1998
`10/1991
`5/1993
`7/1993
`8/1993
`12/1993
`1/1995
`3/1995
`6/1998
`5/1999
`
`(List continued on next page.)
`
`OTHER PUBLICATIONS
`
`Wantanabe et al., ASCO, Vol. 17, abstract 702, May 15-18,
`1998 Annual Meeting.*
`
`(List continued on next page.)
`
`Primary Examiner-Anthony C. Caputa
`Assistant Examiner-Anne L. Holleran
`(74) Attorney, Agent, or Firm-Wendy M. Lee
`
`(57)
`
`ABSTRACT
`
`The present invention concerns the treatment of disorders
`characterized by the overexpression of ErbB2. More
`specifically, the invention concerns the treatment of human
`patients susceptible to or diagnosed with cancer overex(cid:173)
`pressing ErbB2 with anti-ErbB2 antibody.
`
`33 Claims, 5 Drawing Sheets
`
`GNE-HER_000657455
`
`

`

`Case 1:18-cv-00924-CFC Document 312-1 Filed 07/19/19 Page 3 of 287 PageID #: 23800
`
`US 6,627,196 Bl
`
`1
`DOSAGES FOR TREATMENT WITH ANTI(cid:173)
`ERBB2 ANTIBODIES
`
`RELATED APPLICATIONS
`
`This application is a non-provisional application filed
`under 37 CFR 1.53(b)(l), claiming priority under 35 USC
`119(e) to provisional application No. 60/151,018, filed Aug.
`27, 1999 and No. 60/213,822, filed Jun. 23, 2000, the
`contents of which are incorporated herein by reference.
`
`FIELD OF THE INVENTION
`
`10
`
`The present invention concerns the treatment of disorders
`characterized by the overexpression of ErbB2 or disorders
`expressing epidermal growth factor receptor (EGFR), com(cid:173)
`prising administering to a human or animal presenting the
`disorders a therapeutically effective amount of an antibody
`that binds ErbB2. More specifically, the invention concerns
`the treatment of human patients susceptible to or diagnosed
`with cancer overexpressing ErbB2 or expressing EGFR, 20
`where the treatment is with an anti-ErbB2 antibody admin(cid:173)
`istered by front loading the dose of antibody during treat(cid:173)
`ment by intravenous and/or subcutaneous administration.
`The invention optionally includes treatment of cancer in a
`human patient with a combination of an anti-ErbB2 antibody
`and a chemotherapeutic agent, such as, but not limited to, a
`taxoid. The taxoid may be, but is not limited to paclitaxel or
`docetaxel. The invention further includes treatment of can(cid:173)
`cer in a human patient with a combination of anti-ErbB2
`antibody and a chemotherapeutic agent, such as, but not
`limited to, an anthracycline derivative. Optionally, treatment
`with a combination of anti-ErbB2 and an anthracycline
`derivative includes treatment with an effective amount of a
`cardioprotectant. The present invention further concerns
`infrequent dosing of anti-ErbB2 antibodies.
`
`2
`inhibit the tumorigenic growth of neu-transformed NIH-3T3
`cells as well as rat neuroblastoma cells (from which the neu
`oncogene was initially isolated) implanted into nude mice.
`Drebin et al. in Oncogene 2:387-394 (1988) discuss the
`5 production of a panel of antibodies against the rat neu gene
`product. All of the antibodies were found to exert a cyto(cid:173)
`static effect on the growth of neu-transformed cells sus(cid:173)
`pended in soft agar. Antibodies of the IgM, IgG2a and IgG2b
`isotypes were able to mediate significant in vitro lysis of
`neu-transformed cells in the presence of complement,
`whereas none of the antibodies were able to mediate high
`levels of antibody-dependent cellular cytotoxicity (ADCC)
`of the neu-transformed cells. Drebin et al. Oncogene
`2:273-277 (1988) report that mixtures of antibodies reactive
`with two distinct regions on the p185 molecule result in
`l5 synergistic anti-tumor effects on neu-transformed NIH-3T3
`cells implanted into nude mice. Biological effects of anti-neu
`antibodies are reviewed in Myers et al., Meth. Enzym.
`198:277-290 (1991). See also W094/22478 published Oct.
`13, 1994.
`Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989)
`describe the generation of a panel of anti-ErbB2 antibodies
`which were characterized using the human breast tumor cell
`line SKBR3. Relative cell proliferation of the SKBR3 cells
`following exposure to the antibodies was determined by
`25 crystal violet staining of the monolayers after 72 hours.
`Using this assay, maximum inhibition was obtained with the
`antibody called 4D5 which inhibited cellular proliferation by
`56%. Other antibodies in the panel,including 7C2 and 7F3,
`reduced cellular proliferation to a lesser extent in this assay.
`30 Hudziak et al. conclude that the effect of the 4D5 antibody
`on SKBR3 cells was cytostatic rather than cytotoxic, since
`SKBR3 cells resumed growth at a nearly normal rate fol(cid:173)
`lowing removal of the antibody from the medium. The
`antibody 4D5 was further found to sensitize p 185
`35 -overexpressing breast tumor cell lines to the cytotoxic
`effects of TNF-a. See also W089/06692 published Jul. 27,
`1989. The anti-ErbB2 antibodies discussed in Hudziak et al.
`are further characterized in Fendly et al. Cancer Research
`50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A
`40 (1990); Sarup et al. Growth Regulation 1:72---82 (1991);
`Shepard et al. J. Clin. Immunol. 11(3):117-127 (1991);
`Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis
`et al. Cancer Immunol. Immunother. 37:255-263 (1993);
`Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al.
`45 Cancer Research 54:5301-5309 (1994); Sliwkowski et al.J.
`Biol. Chem. 269(20): 14661-14665 (1994); Scott et al. J.
`Biol. Chem. 266:14300---5 (1991); and D'souza et al. Proc.
`Natl. Acad. Sci.91:7202-7206 (1994).
`Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe
`50 two antibodies which were selected for their reactivity on
`the lung adenocarcinoma cell line (Calu-3) which overex(cid:173)
`presses ErbB2. One of the antibodies, called MGR3, was
`found to internalize, induce phosphorylation of ErbB2, and
`inhibit tumor cell growth in vitro.
`McKenzie et al. Oncogene 4:543-548 (1989) generated a
`panel of anti-ErbB2 antibodies with varying epitope
`specificities, including the antibody designated TAl. This
`TAl antibody was found to induce accelerated endocytosis
`of ErbB2 (see Maier et al. Cancer Res. 51:5361-5369
`60 [1991]). Bacus et al. Molecular Carcinogenesis 3:350---362
`(1990) reported that the TAl antibody induced maturation of
`the breast cancer cell linesAU-565 (which overexpresses the
`erbB2 gene) and MCF-7 (which does not). Inhibition of
`growth and acquisition of a mature phenotype in these cells
`65 was found to be associated with reduced levels of ErbB2
`receptor at the cell surface and transient increased levels in
`the cytoplasm.
`
`BACKGROUND OF THE INVENTION
`
`Proto-oncogenes that encode growth factors and growth
`factor receptors have been identified to play important roles
`in the pathogenesis of various human malignancies, includ(cid:173)
`ing breast cancer. It has been found that the human ErbB2
`gene (erbB2, also known as her2, or c-erbB-2), which
`encodes a 185-kd transmembrane glycoprotein receptor
`(p185HER2
`) related to the epidermal growth factor receptor
`(EGFR), is overexpressed in about 25% to 30% of human
`breast cancer (Slamon et al., Science 235:177-182 [1987];
`Slamon et al., Science 244:707-712 [1989]).
`Several lines of evidence support a direct role for ErbB2
`in the pathogenesis and clinical aggressiveness of ErbB2-
`overexpressing tumors. The introduction of ErbB2 into
`non-neoplastic cells has been shown to cause their malignant
`transformation (Hudziak et al., Proc. Natl. Acad. Sci. USA
`84:7159-7163 [1987]; DiFiore et al., Science 237:78-182
`[1987]). Transgenic mice that express HER2 were found to 55
`develop mammary tumors (Guy et al.,Proc. Natl.Acad. Sci.
`USA 89:10578-10582 [1992]).
`Antibodies directed against human erbB2 protein prod(cid:173)
`ucts and proteins encoded by the rat equivalent of the erbB2
`gene (neu) have been described. Drebin et al., Cell
`41:695-706 (1985) refer to an IgG2a monoclonal antibody
`which is directed against the rat neu gene product. This
`antibody called 7.16.4 causes down-modulation of cell sur(cid:173)
`face p185 expression on B104-1-1 cells (NIH-3T3 cells
`transfected with the neu pro to-oncogene) a inhibits colony
`formation of these cells. In Drebin et al. PNAS (USA)
`83:9129-9133 (1986), the 7.16.4 antibody was shown to
`
`GNE-HER_000657464
`
`

`

`Case 1:18-cv-00924-CFC Document 312-1 Filed 07/19/19 Page 4 of 287 PageID #: 23801
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`US 6,627,196 Bl
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`5
`administered is sufficient to maintain the target trough serum
`concentration such that the interval between administration
`cycles is at least one week. Preferably the trough serum
`concentration does not exceed 2500 µg!ml and does not fall
`below 0.01 µg!ml during treatment. The front loading drug
`treatment method of the invention has the advantage of
`increased efficacy by reaching a target serum drug concen(cid:173)
`tration early in treatment. The subcutaneous delivery of
`maintenance doses according to the invention has the advan(cid:173)
`tage of being convenient for the patient and health care 10
`professionals, reducing time and costs for drug treatment.
`Preferably, the initial dose (or the last dose within an initial
`dose series) is separated in time from the first subsequent
`dose by 4 weeks or less, preferably 3 weeks or less, more
`preferably 3 weeks or less, most preferably 1 week or less. 15
`In an embodiment of the invention, the initial dose of
`anti-ErbB2 is 6 mg/kg, 8 mg/kg, or 12 mg/kg delivered by
`intravenous or subcutaneous administration, such as intra(cid:173)
`venous infusion or subcutaneous bolus injection. The sub(cid:173)
`sequent maintenance doses are 2 mg/kg delivered once per 20
`week by intravenous infusion, intravenous bolus injection,
`subcutaneous infusion, or subcutaneous bolus injection. The
`choice of delivery method for the initial and maintenance
`doses is made according to the ability of the animal or
`human patient to tolerate introduction of the antibody into 25
`the body. Where the antibody is well-tolerated, the time of
`infusion may be reduced. The choice of delivery method as
`disclosed for this embodiment applies to all drug delivery
`regimens contemplated according to the invention.
`In another embodiment, the invention includes an initial
`dose of 12 mg/kg anti-ErbB2 antibody, followed by subse(cid:173)
`quent maintenance doses of 6 mg/kg once per 3 weeks.
`In still another embodiment, the invention includes an
`initial dose of 8 mg/kg anti-ErbB2 antibody, followed by 6
`mg/kg once per 3 weeks.
`In yet another embodiment, the invention includes an
`initial dose of 8 mg/kg anti-ErbB2 antibody, followed by
`subsequent maintenance doses ofS mg/kg once per week or
`8 mg/kg once every 2 to 3 weeks.
`In another embodiment, the invention includes initial
`doses of at least 1 mg/kg, preferably 4 mg/kg, anti-ErbB2
`antibody on each of days 1, 2 and 3, followed by subsequent
`maintenance doses of 6 mg/kg once per 3 weeks.
`In another embodiment, the invention includes an initial
`dose of 4 mg/kg anti-ErbB2 antibody, followed by subse(cid:173)
`quent maintenance doses of 2 mg/kg twice per week,
`wherein the maintenance doses are separated by 3 days.
`In still another embodiment, the invention includes a
`cycle of dosing in which delivery of anti-ErbB2 antibody is
`2-3 times per week for 3 weeks. In one embodiment of the
`invention, each dose is approximately 25 mg/kg or less for
`a human patient, preferably approximately 10 mg/kg or less.
`This 3 week cycle is preferably repeated as necessary to
`achieve suppression of disease symptoms.
`In another embodiment, the invention includes a cycle of
`dosing in which delivery of anti-ErbB2 antibody is daily for
`5 days. According to the invention, the cycle is preferably
`repeated as necessary to achieve suppression of disease
`symptoms.
`The disorder preferably is a benign or malignant tumor
`characterized by the overexpression of the ErbB2 receptor,
`e.g. a cancer, such as, breast cancer, squamous cell cancer,
`small-cell lung cancer, non-small cell lung cancer, gas(cid:173)
`trointestinal cancer, pancreatic cancer, glioblastoma, cervi(cid:173)
`cal cancer, ovarian cancer, liver cancer, bladder cancer,
`hepatoma, colon cancer, colorectal cancer, endometrial
`
`6
`carcinoma, salivary gland carcinoma, kidney cancer, liver
`cancer, prostate cancer, vulva! cancer, thyroid cancer,
`hepatic carcinoma and various types of head and neck
`cancer. The method of the invention may further comprise
`5 administration of a chemotherapeutic agent other than an
`anthracycline, e.g. doxorubicin or epirubicin. The chemo(cid:173)
`therapeutic agent preferably is a taxoid, such as TAXOL®
`(paclitaxel) or a TAXOL® derivative.
`Preferred anti-ErbB2 antibodies bind the extracellular
`domain of the ErbB2 receptor, and preferably bind o the
`epitope 4D5 or 3H4 within the ErbB2 extracellular domain
`sequence. More preferably, the antibody is the antibody
`4D5, most preferably in a humanized form. Other preferred
`ErbB2-binding antibodies include, but are not limited to,
`antibodies 7C2, 7F3, and 2C4, preferably in a humanized
`form.
`The method of the present invention is particularly suit(cid:173)
`able for the treatment of breast or ovarian cancer, charac(cid:173)
`terized by the overexpression of the ErbB2 receptor.
`The present application also provides a method of therapy
`involving infrequent dosing of an anti-ErbB2 antibody. In
`particular, the invention provides a method for the treatment
`of cancer ( e.g. cancer characterized by overexpression of the
`ErbB2 receptor) in a human patient comprising administer(cid:173)
`ing to the patient a first dose of an anti-ErbB2 antibody
`followed by at least one subsequent dose of the antibody,
`wherein the first dose and subsequent dose are separated
`from each other in time by at least about two weeks (e.g.
`from about two weeks to about two months), and optionally
`30 at least about three weeks (e.g. from about three weeks to
`about six weeks). For instance, the antibody may be admin(cid:173)
`istered about every three weeks, about two to about 20 times,
`e.g. about six times. The first dose and subsequent dose may
`each be from about 2 mg/kg to about 16 mg/kg; e.g. from
`35 about 4 mg/kg to about 12 mg/kg; and optionally from about
`6 mg/kg to about 12 mg/kg. Generally, two or more subse(cid:173)
`quent doses (e.g. from about two to about ten subsequent
`doses) of the antibody are administered to the patient, and
`those subsequent doses are preferably separated from each
`40 other in time by at least about two weeks (e.g. from about
`two weeks to about two months), and optionally at least
`about three weeks (e.g. from about three weeks to about six
`weeks). The two or more subsequent doses may each be
`from about 2 mg/kg to about 16 mg/kg; or from about 4
`45 mg/kg to about 12 mg/kg; or from about 6 mg/kg to about
`12 mg/kg. The invention additionally provides an article of
`manufacture, comprising a container, a composition within
`the container comprising an anti-ErbB2 antibody, and a
`package insert containing instructions to administer the
`50 antibody according to such methods.
`The presently described dosing protocols may be applied
`to other anti-ErbB antibodies such as anti-epidermal growth
`factor receptor (EGFR), anti-ErbB3 and anti-ErbB4 anti(cid:173)
`bodies. Thus, the invention provides a method for the
`55 treatment of cancer in a human patient, comprising admin(cid:173)
`istering an effective amount of an anti-ErbB antibody to the
`human patient, the method comprising administering to the
`patient an initial dose of at least approximately 5 mg/kg of
`the anti-ErbB antibody; and administering to the patient a
`60 plurality of subsequent doses of the antibody in an amount
`that is approximately the same or less than the initial dose.
`Alternatively, or additionally, the invention pertains to a
`method for the treatment of cancer in a human patient
`comprising administering to the patient a first dose of an
`65 anti-ErbB antibody followed by at least one subsequent dose
`of the antibody, wherein the first dose and subsequent dose
`are separated from each other in time by at least about two
`
`GNE-HER_000657466
`
`

`

`Case 1:18-cv-00924-CFC Document 312-1 Filed 07/19/19 Page 5 of 287 PageID #: 23802
`
`US 6,627,196 Bl
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`8
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`7
`weeks. The invention additionally provides an article of
`manufacture, comprising a container, a composition within
`the container comprising an anti-ErbB antibody, and a
`package insert containing instructions to administer the
`antibody according to such methods.
`In another aspect, the invention concerns an article of
`manufacture, comprising a container, a composition within
`the container comprising an anti-ErbB2 antibody, optionally
`a label on or associated with the container that indicates that
`the composition can be used for treating a condition char(cid:173)
`acterized by overexpression of ErbB2 receptor, and a pack(cid:173)
`age insert containing instructions to avoid the use of
`anthracycline-type chemotherapeutics in combination with
`the composition. According to the invention, the package
`insert further includes instructions to administer the anti(cid:173)
`ErbB2 antibody at an initial dose of 5 mg/kg followed by the
`same or smaller subsequent dose or doses. In another
`embodiment of the invention, the package insert further
`includes instructions to administer the anti-ErbB2 antibody
`subcutaneously for at least one of the doses, preferably for
`all of the subsequent doses following the initial dose, most
`preferably for all doses.
`In a further aspect, the invention provides a method of
`treating ErbB2 expressing cancer in a human patient com(cid:173)
`prising administering to the patient effective amounts of an
`anti-ErbB2 antibody and a chemotherapeutic agent. In one
`embodiment of the invention, the chemotherapeutic agent is
`a taxoid including, but not limited to, paclitaxel and doc(cid:173)
`etaxel. In another embodiment, the chemotherapeutic agent
`is an anthracyline derivative including, but not limited to,
`doxorubicin or epirubicin. In still another embodiment of the 30
`invention, treatment with an anti-ErbB2 antibody and an
`anthracycline derivative further includes administration of a
`cardioprotectant to the patient. In still another embodiment,
`an anthracycline derivative is not administered to the patient
`with the anti-ErbB2 antibody. One or more additional che- 35
`motherapeutic agents may also be administered to the
`patient. The cancer is preferably characterized by overex(cid:173)
`pression of ErbB2.
`The invention further provides an article of manufacture
`comprising a container, a composition within the container 40
`comprising an anti-ErbB2 antibody and a package insert
`instructing the user of the composition to administer the
`anti-ErbB2 antibody composition and a chemotherapeutic
`agent to a patient. In another embodiment, the chemothera(cid:173)
`peutic agent is other than an anthracycline, and is preferably 45
`a taxoid, such as TAXOL®. In still another embodiment, the
`chemotherapeutic agent is an anthracycline, including but
`not limited to, doxorubicin or epirubicin. In yet another
`embodiment, the chemotherapeutic agent is an anthracycline
`and the package insert further instructs the user to administer 50
`a cardioprotectant.
`The methods and compositions of the invention comprise
`an anti-ErbB2 antibody and include a humanized anti-ErbB2
`antibody. Thus, the invention further pertains to a compo(cid:173)
`sition comprising an antibody that binds ErbB2 and the use 55
`of the antibody for treating ErbB2 expressing cancer, e.g.,
`ErbB2 overexpressing cancer, in a human. The invention
`also pertains to the use of the antibody for treating EGFR
`expressing cancer. Preferably the antibody is a monoclonal
`antibody 4D5, e.g., humanized 4D5 (and preferably 60
`huMAb4D5-8 (HERCEPTIN® anti-ErbB2 antibody); or
`monoclonal antibody 2C4, e.g., humanized 2C4. The anti(cid:173)
`body may be an intact antibody (e.g., an intact IgG,
`antibody) or an antibody fragment (e.g., a Fab, F(ab')2 ,
`diabody, and the like). The variable light chain and variable 65
`heavy chain regions of humanized anti-ErbB2 antibody 2C4
`are shown in FIGS. SA and SB.
`
`FIG. 1 shows epitope-mapping of the extracellular
`domain of ErbB2 as determined by truncation mutant analy(cid:173)
`sis and site-directed mutagenesis (Nakamura et al. J. of
`5 Virology 67 (10):6179-6191 [October 1993]; Renz etJ. Cell
`Biol. 125(6):1395-1406 [June 1994]). The anti-proliferative
`MAbs 4D5 and 3H4 b bind adjacent to the transmembrane
`domain. The various ErbB2-ECD truncations or point muta(cid:173)
`tions were prepared from cDNA using polymerase chain
`10 reaction technology. The ErbB2 mutants were expressed as
`gD fusion proteins in a mammalian expression plasmid. This
`expression plasmid uses the cytomegalovirus promoter/
`enhancer with SV40 termination and polyadenylation sig(cid:173)
`nals located downstream of the inserted cDNA. Plasmid
`15 DNA was transfected into 293S cells. One day following
`transfection, the cells were metabolically labeled overnight
`in methionine and cysteine-free, low glucose DMEM con(cid:173)
`taining 1 % dialyzed fetal bovine serum and 25 µCi each of
`35S methionine and 35S cysteine. Supernatants were har-
`20 vested either the ErbB2 MAbs or control antibodies were
`added to the supernatant and incubated 2-4 hours at 4 ° C.
`The complexes were precipitated, applied to a 10-20%
`Tricine SDS gradient gel and electrophoresed at 100 V. The
`gel was electroblotted onto a membrane and analyzed by
`25 autoradiography. SEQ ID NOs:8 and 9 depict the 3H4 and
`4D5 epitopes, respectively.
`FIG. 2 depicts with underlining the amino acid sequence
`of Domain 1 of ErbB2 (SEQ ID NO: 1). Bold amino acids
`indicate the location of the epitope recognized by MAbs 7C2
`and 7F3 as determined by deletion mapping, i.e. the "7C2/
`7F3 epitope" (SEQ ID N0:2).
`FIG. 3 is a graph of anti-ErbB2 antibody
`(HERCEPTIN®) trough serum concentration (ug/ml, mean
`±SE, dark circles) by week from week 2 through week 36 for
`ErbB2 overexpressing patients treated with HERCEPTIN®
`anti-ErbB2 antibody at 4 mg/kg initial dose, followed by 2
`mg/kg weekly. The number of patients at each time point is
`represented by "n" (white squares).
`FIG. 4A is a linear plot of tumor volume changes over
`time in mice treated with HERCEPTIN® anti-ErbB2 anti(cid:173)
`body. FIG. 4B is a semi-logarithmic plot of the same data as
`in FIG. 4A such that the variation in tumor volume for the
`treated animals is observed more readily.
`FIGS. SA and SB depict alignments of the amino acid
`sequences of the variable light (VL)(FIG. SA) and variable
`heavy (V H) (FIG. SB) domains of murine monoclonal anti(cid:173)
`body 2C4 (SEQ ID Nos. 10 and 11, respectively); VL and V H
`domains of humanized Fab version 574 (SEQ ID Nos. 12
`and 13, respectively), and human VL and V H consensus
`frameworks (hum Kl, light kappa subgroup I; humIII, heavy
`subgroup III) (SEQ ID Nos. 14 and 15, respectively).
`Asterisks identify differences between humanized Fab ver(cid:173)
`sion 574 and murine monoclonal antibody 2C4 or between
`humanized Fab version 574 and the human framework.
`Complementarity Determining Regions (CDRs) are in
`brackets. Humanized Fab version 574, with the changes
`ArgH71 Val, AspH73Arg and IleH69Leu, appears to have
`binding restored to that of the original chimeric 2C4 Fab
`fragment. Additional FR and/or CDR residues, such as L2,
`L54, L55, L56, H35 and/or H48, may be modified (e .g.
`substituted as follows-IleL2Thr; ArgL54Leu; TyrL55Glu;
`ThrL56Ser; AspH35Ser; and ValH48Ile) in order to further
`refine or enhance binding of the humanized antibody.
`Alternatively, or additionally, the humanized antibody may
`be affinity matured in order to further improve or refine its
`affinity and/or other biological activities.
`
`GNE-HER_000657467
`
`

`

`Case 1:18-cv-00924-CFC Document 312-1 Filed 07/19/19 Page 6 of 287 PageID #: 23803
`
`US 6,627,196 Bl
`
`57
`the subsequent doses are separated in time from each
`other by at least two weeks.
`2. The method of claim 1, wherein the initial dose is at
`least approximately 6 mg/kg.
`3. The method of claim 2, wherein the initial dose is at
`least approximately 8 mg/kg.
`4. The method of claim 3, wherein the initial dose is at
`least approximately 12 mg/kg.
`5. The method of claim 1, wherein the subsequent doses
`are separated in time from each other by at least three weeks.
`6. The method of claim 1, wherein the initial dose is
`administered by intravenous injection, and wherein at least
`one subsequent dose is administered by subcutaneous injec(cid:173)
`tion.
`7. The method of claim 1, wherein the initial dose is 15
`administered by intravenous injection, wherein at least two
`subsequent doses are administered, and wherein each sub(cid:173)
`sequent dose is administered by a method selected from the
`group consisting of intravenous injection and subcutaneous
`injection.
`8. The method of claim 1, wherein the initial dose and at
`least one subsequent dose are administered by subcutaneous
`injection.
`9. The method of claim 1, wherein the initial dose is
`selected from the group consisting of approximately 6 25
`mg/kg, 8 mg/kg, or 12 mg/kg, wherein the plurality of
`subsequent doses are at least approximately 2 mg/kg.
`10. The method of claim 9, wherein the plurality of
`subsequent doses are separated in time from each other by
`at least three weeks.
`11. The method of claim 10, wherein the initial dose is
`approximately 8 mg/kg, and wherein at least one subsequent
`dose is approximately 6 mg/kg.
`12. The method of claim 10, wherein the initial dose is
`approximately 12 mg/kg, and wherein at least one subse- 35
`quent dose is approximately 6 mg/kg.
`13. The method of claim 9, wherein the initial dose is
`approximately 8 mg/kg, and wherein at least one subsequent
`dose is approximately 8 mg/kg.
`14. The method of claim 9, wherein the initial dose is 40
`approximately 8 mg/kg, wherein at least one subsequent
`dose is 8 mg/kg, and wherein administration of the initial
`dose and subsequent doses are separated in time by at least
`2 weeks.
`15. The method of claim 14, wherein the initial dose and 45
`subsequent doses are separated in time by at least 3 weeks.
`16. A method for the treatment of a human patient
`diagnosed with cancer characterized by overexpression of
`ErbB2 receptor, comprising administering an effective
`amount of an anti-ErbB2 antibody to the human patient, the 50
`method comprising:
`administering to the patient an initial dose of the antibody,
`wherein the initial dose is a plurality of doses, wherein
`each of the plurality of initial doses is at least approxi(cid:173)
`mately 1 mg/kg and is administered on at least 3 55
`consecutive days, and administering to the patient at
`least 1 subsequent dose of the antibody, wherein at least
`one subsequent dose is at least approximately 6 mg/kg,
`and wherein administration of the last initial dose and
`
`58
`the first subsequent and additional subsequent doses are
`separated in time by at least 3 weeks.
`17. The method of claim 1, wherein said cancer is selected
`from the group consisting of breast cancer, leukemia, squa-
`5 mous cell cancer, small-cell lung cancer, non-small cell lung
`cancer, gastrointestinal cancer, pancreatic cancer,
`glioblastoma, cervical cancer, ovarian cancer, liver cancer,
`bladder cancer, hepatoma, colon cancer, colorectal cancer,
`endometrial carcinoma, salivary gland carcinoma, kidney
`cancer, liver cancer, prostate cancer, vulva! cancer, thyroid
`10 cancer, hepatic carcinoma and various types of head and
`neck cancer.
`18. The method of claim 17, wherein said cancer is breast
`cancer.
`19. The method of claim 18, wherein said cancer is
`metastatic breast carcinoma.
`20. The method of claim 1, wherein said antibody binds
`to the extracellular domain of the ErbB2 receptor.
`21. The method of claim 20, wherein said antibody binds
`to epitope 4D5 within the ErbB2 extracellular domain
`20 sequence.
`22. The method of claim 21, wherein said antibody is a
`humanized 4D5 anti-ErbB2 antibody.
`23. The method of claim 1, wherein efficacy is measured
`by determining the time to disease progression or the
`response rate.
`24. A method for the treatment of cancer in a human
`patient comprising administering to the patient a first dose of
`an anti-ErbB2 antibody followed by two or more subsequent
`doses of the antibody, wherein the subsequent doses are
`30 separated in time from each other by at le