Please type a plus sign ( +) inside this box G]
`
`EM 168 882. 385 US; Express MaD Number
`
`December 10 1998: Date of Deposit
`
`~!(?Of)~
`
`Attorney Docket P1256R1
`PATENT
`0 ~
`.-----------------------------------------------------------------------------~~ ~
`0..0"\ ;;;::::::;;
`CERTIFrCATION UNDER 37 CFR 1.10
`.cd:" ~~
`tll...a
`'
`.co=o
`c:::;:)~
`::>C"l ~.-1
`---===='
`o_...
`N
`o::l' ..... ·=.-1
`IJ)c;::) ~
`u ~
`
`I hereby il:ertify that this Ncm-pr.ovisiomd Applicaticm Transmittal and the documtmno r•ferred w u ern:Josed therein are
`being dePQsitecf with 1he United StetelJ Postel Service "Express Mail Post Office to Addressee" service under 37 CFR
`1.10 on the date indicated above and is addresnd to the Aeskltant Commiseioner of Patents. Washington, D.C, 20231.
`
`LJ,iwt.Ltt·
`
`Ann Savelli
`
`t")
`
`:::::::::
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BOX PATENT APPLICATION
`Assistant Commissioner of Patents
`Washington, D.C. 20231
`
`NON-PROVISIONAL APPLICATION TRANSMITTAL UNDER 37 CFR 1.53lbl
`
`Transmitted herewith for filing is a non-provisional patent application:
`
`lnventor(s) (or Application "Identifier"):
`
`Virginia E. Paton
`Steven Shak
`
`Title:
`
`TREATMENT WITH ANTI-ErbB2 ANTIBODIES
`
`..
`
`1 .
`
`I ]
`
`lx ]
`
`I 1
`
`2.
`
`Type of Application
`
`This application is for an original, non-provisional application.
`
`This is a non-provisional application claiming priority to provisional application no.
`, filed
`December 12 1997 , the entire disclosure of which is hereby
`60/069 346
`incorporated by reference.
`
`application
`[ 1 divisional
`[ 1 continuation
`[ 1 continuation-in-part
`This is a
`claiming priority to application Serial Number_, filed _,the entire disclosure of which
`is hereby incorporated by reference.
`
`Papers Enclosed Which Are Required For Filing Date Under 37 CFR 1 .53(b)
`(Non-provisional l
`~ pages of specification
`pages of claims
`page(s) of abstract
`_2_ sheet(s) of drawings
`[x] formal
`[) informal
`
`HOSPIRA EX. 1121
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`P1256Rl
`
`3.
`
`Declaration or Oath
`
`Page 2 of4
`
`(for new and CIP applications; also for Cont./Div. where inventor(s} are being added}
`_x_ An unexecuted declaration of the inventors is enclosed. An executed declaration of the
`inventors will follow.
`
`(for Cont./Div. where inventorship is the same or inventor(s} being deleted}
`A copy of the executed declaration/oath filed in the prior application is enclosed (37
`CFR 1.63(d)).
`
`(for Cont./Div. where inventor(s) being deleted)
`A signed statement is attached deleting inventor(s) named in the prior application (see
`37 CFR 1.63(d)(2) and 1.33(b)).
`
`4.
`
`Assignment
`
`(for new and CIP applications}
`_x_ An Assignment of the invention to GENENTECH, INC. [] is enclosed with attached
`Recordation Form Cover Sheet [x] will follow.
`
`(for cont./div.)
`The prior application is assigned of record to Genentech, Inc.
`
`5.
`
`Amendments
`
`(for continuation and divisional applications)
`
`Cancel in this application original claims_ of the prior application before calculating the
`filing fee. (At least one original independent claim must be retained for filing purposes.)
`
`(Claims added by this amendment have been
`A preliminary amendment is enclosed.
`properly numbered consecutively beginning with the number next following the highest
`numbered original claim in the prior application.)
`
`Relate Back-- 35 U.S.C. 120 or 35 U.S.C. 119
`
`__ Amend the specification by inserting before the first line the sentence:
`
`--This is a
`
`non-provisional application
`continuation
`divisional
`continuation-in-part
`
`of co-pending application(s)
`
`Serial No. _ filed on_, which application(s) is(are) incorporated herein by reference and
`to which application(s) priority is claimed under 35 USC § 1 20. --
`International Application_ filed on_ which designated the U.S .. which application(s)
`is(are) incorporated herein by reference and to which application(s) priority is claimed
`
`HOSPIRA EX. 1121
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`P1256Rl
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`Page 3 of 4
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`under 35 USC § 120.--
`provisional application No. _filed _the entire disclosure of which is hereby incorporated
`by reference and to which application(s) priority is claimed under 35 USC §119.--.
`
`6.
`
`Fee Calculation (37 CFR 1.16)
`The fee has
`
`Total
`Claims
`
`Independent
`Claims
`
`19
`
`2
`
`- 20 =
`
`- 3=
`
`0
`
`0
`
`X $18.00
`
`X $78.00
`
`Multiple dependent claim(s), if any
`
`+ $260.00
`
`Filing Fee Calculation
`
`$0.00
`
`$0.00
`
`$0.00
`
`$760.00
`
`7.
`
`8.
`
`9.
`
`Method of Payment of Fees
`The Commissioner is hereby authorized to charge Deposit Account No. 07-0630 in the amount of
`$760.00. A duplicate coPY of this transmittal is enclosed.
`
`Authorization to Charge Additional Fees
`The Commissioner is hereby authorized to charge any additional fees required under 37 CFR § 1.16
`and 1.17, or credit overpayment to Deposit Account No. 07-0630. A duplicate copy of this sheet
`js enclosed.
`
`Additional Papers Enclosed
`Information Disclosure Statement (37 CFR § 1.98) w/ PT0-1449 and citations
`[]
`[x] Submission of "Sequence Listing", computer readable copy, certificate re: sequence listing,
`and/or amendment pertaining thereto for biological invention containing nucleotide and/or
`amino acid sequence.
`[] A new Power of Attorney or authorization of agent.
`[] Other:
`
`10.
`
`(for continuation and divisional applications)
`Maintenance of Copendency of Prior Application
`[This item must be completed and the necessary papers filed in the prior application if the period
`set in the prior application has run]
`
`A petition, fee and/or response has been filed to extend the term in the pending
`prior application until
`A copy of the petition for extension of time in the prior application is attached.
`
`HOSPIRA EX. 1121
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`P1256Rl
`
`Page 4 of4
`
`11.
`
`Correspondence Address:
`
`_x_
`
`Address all future communications to:
`
`GENENTECH, INC.
`Attn: Wendy M. Lee
`1 DNA Way
`South San Francisco, CA
`(650) 225-1994
`
`94080-4990
`
`Respectfully submitted,
`GENENTECH, INC.
`
`By: J~ldov
`
`W611'aY M. Lee
`Reg. No. 40,378
`
`Date: December 10, 1998
`
`1 DNA Way
`So. San Francisco, CA 94080-4990
`Phone: (650) 225-1994
`Fax: (650) 952-9881
`
`HOSPIRA EX. 1121
`Page 4
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`

`

`Our Docket No. P1256R1
`EXPRESS MAIL NO: EM 168 882 385 US
`MAILED: December 10, 1998
`
`5
`
`10
`
`TREATMENT WITH ANTI-ErbB2 ANTffiODIES
`
`This is a non-provisional application claiming priority to provisional application no.
`
`60/069,346, filed December 12, 1997, the entire disclosure of which is hereby incorporated by
`
`reference.
`Field of the Invention
`
`The present invention concerns the treatment of disorders characterized by the
`
`overexpression ofErbB2. More specifically, the invention concerns the treatment of human
`
`patients susceptible to or diagnosed with cancer overexpressing ErbB2 with a combination of an
`
`anti-ErbB2 antibody and a chemotherapeutic agent other than an anthracycline, e.g. doxorubicin
`
`or epirubicin.
`
`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, including
`
`2Gk
`~= ...
`
`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 (p 185HER2) 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]).
`
`25
`
`Several lines of evidence support a direct role for ErbB2 in the pathogenesis and clinical
`
`aggressiveness ofErbB2-overexpressing tumors. The introduction ofErbB2 into non-neoplastic
`
`cells has been shown to cause their malignant transformation (Hudziak et al., Proc. Nat/. A cad.
`
`Sci. USA 84:7159-7163 [1987];DiFioreeta/.,Science237:78-182 [1987]). Transgenicmicethat
`
`express HER2 were found to develop mammary tumors (Guy eta!., Proc. Nat!. A cad Sci. USA
`
`1
`
`HOSPIRA EX. 1121
`Page 5
`
`

`

`89:10578-10582 [1992]).
`
`Antibodies directed against human erbB2 protein products and proteins encoded by the rat
`
`equivalent ofthe erbB2 gene (neu) have been described. Drebin et al., Cell41:695-706 (1985)
`
`refer to an IgG2a monoclonal antibody which is directed against the rat neu gene product. This
`
`5
`
`antibody called 7.16.4 causes down-modulation of cell surface p185 expression on B104-1-1 cells
`
`(NIH~3T3 cells transfected with the neu proto-oncogene) and inhibits colony formation of these
`In Drebin et al. PNAS (USA) 83:9129-9133 (1986), the 7.16.4 antibody was shown to
`
`cells.
`
`inhibit the tumorigenic growth ofneu-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 production of a panel of antibodies against the rat neu
`
`10
`
`gene product. All of the antibodies were found to exert a cytostatic effect on the growth of neu(cid:173)
`
`transformed cells suspended 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 p 185 molecule result in
`
`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 October 13, 1994.
`
`Hudziak et al., Mol. Cell. Bioi. 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 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%.
`
`25
`
`Other antibodies in the panel, including 7C2 and 7F3, reduced cellular proliferation to a lesser
`
`extent in this assay. 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
`
`following removal of the antibody from the medium. The antibody 4D5 was further found to
`
`sensitize p185erbB2-overexpressing breast tumor cell lines to the cytotoxic effects ofTNF-a. See
`
`2
`
`HOSPIRA EX. 1121
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`

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`also W089/06692 published July 27, 1989. The anti-ErbB2 antibodies discussed in Hudziak et
`
`ai. are further characterized in Fendly et al. Cancer Research 50:1550-1558 (1990); Kotts et ai.
`
`In Vitro 26(3):59A (1990); Sarup et al. Growth Regulation l :72-82 (1991); Shepard et al. J.
`
`Clin. Irnrnunol. 11(3):117-127 (1991); Kumar eta!. Mol. Cell. Bioi. 11(2):979-986 (1991); Lewis
`
`5
`
`et al. Cancer Immunol. Immunother. 37:255-263 (1993); Pietras et al. Oncogene 9:1829-1838
`
`(1994); Vitetta et al. Cancer Research 54:5301-5309 (1994); Sliwkowski eta!. J. Bioi. Chern.
`
`269(20): 14661-14665 (1994); Scott et ai. J. Bioi. Chern. 266:14300-5 (1991); and D'souza eta!.
`
`Proc. Natl. Acad Sci. 91:7202-7206 (1994).
`Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe two antibodies which were
`
`10
`
`selected for their reactivity on the lung adenocarcinoma cell line (Calu-3) which overexpresses
`
`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 ofErbB2 (see Maier et al. Cancer Res. 51:5361-5369
`
`[1991]). Bacus et al. Molecular Carcinogenesis 3:350-362 (1990) reported that the TAl
`
`antibody induced maturation of the breast cancer cell lines AU-565 (which overexpresses the
`
`erbB2 gene) and MCF-7 (which does not). Inhibition of growth and acquisition of a mature
`
`phenotype in these cells was found to be associated with reduced levels ofErbB2 receptor at the
`
`cell surface and transient increased levels in the cytoplasm.
`
`Stancovski et ai. PNAS (USA) 88:8691-8695 (1991) generated a panel of anti-ErbB2
`
`antibodies, injected them i.p. into nude mice and evaluated their effect on tumor growth of murine
`fibroblasts transformed by overexpression of the erbB2 gene. Various levels of tumor inhibition
`
`were detected for four of the antibodies, but one of the antibodies (N28) consistently stimulated
`
`25
`
`tumor growth. Monoclonal antibody N28 induced significant phosphorylation of the ErbB2
`
`receptor, whereas the other four antibodies generally displayed low or no phosphorylation(cid:173)
`
`inducing activity. The effect of the anti-ErbB2 antibodies on proliferation of SKBR3 cells was
`
`also assessed. In this SKBR3 cell proliferation assay, two of the antibodies (N12 and N29)
`
`caused a reduction in cell proliferation relative to control. The ability of the various antibodies to
`
`3
`
`HOSPIRA EX. 1121
`Page 7
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`

`

`induce cell lysis in vitro via complement-dependent cytotoxicity (CDC) and antibody-mediated
`
`cell-dependent cytotoxicity (ADCC) was assessed, with the authors of this paper concluding that
`
`the inhibitory function of the antibodies was not attributed significantly to CDC or ADCC.
`
`Bacus et al. Cancer Research 52:2580-2589 (1992) further characterized the antibodies
`
`5
`
`described in Bacus et al. (1990) and Stancovski et al. of the preceding paragraphs. Extending the
`
`i.p. studies of Stancovski et al., the effect of the antibodies after i.v. injection into nude mice
`
`harboring mouse fibroblasts overexpressing human ErbB2 was assessed. As observed in their
`
`earlier work, N28 accelerated tumor growth whereas N12 and N29 significantly inhibited growth
`
`of the ErbB2-expressing cells. Partial tumor inhibition was also observed with the N24 antibody.
`
`10
`
`Bacus et al. also tested the ability of the antibodies to promote a mature phenotype in the human
`
`breast cancer cell lines AU-565 and MDA-MB453 (which overexpress ErbB2) as well as MCF-7
`
`(containing low levels of the receptor). Bacus et al. saw a correlation between tumor inhibition in
`
`vivo and cellular differentiation; the tumor-stimulatory antibody N28 had no effect on
`
`differentiation, and the tumor inhibitory action of the N12, N29 and N24 antibodies correlated
`
`with the extent of differentiation they induced.
`
`Xu etal. Int. J. Cancer 53:401-408 (1993) evaluated a panel ofanti-ErbB2 antibodies for
`
`their epitope binding specificities, as well as their ability to inhibit anchorage-independent and
`
`anchorage-dependent growth of SKBR3 cells (by individual antibodies and in combinations),
`
`modulate cell-surface ErbB2, and inhibit ligand stimulated anchorage-independent growth. See
`also W094/00136 published Jan 6, 1994 and Kasprzyk et al. Cancer Research 52:2771-2776
`(1992) concerning anti-ErbB2 antibody combinations. Other anti-ErbB2 antibodies are discussed
`
`in Hancock eta!. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res. 54:1367-1373
`
`(1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); and Harwerth et al. J. Bioi. Chern.
`
`267:15160-15167 (1992).
`
`:t
`
`~
`
`2~ ~d
`
`25
`
`A recombinant humanized anti-ErbB2 monoclonal antibody (a humanized version of the
`
`murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or HERCEPTJN®) has been
`
`clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received
`
`extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oneal. 14:737-744 [1996]).
`
`ErbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially
`
`4
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`HOSPIRA EX. 1121
`Page 8
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`in patients with primary disease that involves axillary lymph nodes (Slamon et al., [1987] and
`
`[1989], suvra; Ravdin and Chamness, Gene 159:19-27 [1995]; and Hynes and Stem, Biochim
`
`Biophys Acta 1198:165-184 [1994]), and has been linked to sensitivity and/or resistance to
`
`hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide,
`
`5
`
`methotrexate, and fluoruracil) and anthracyclines (Baselga et al., Oncology 11(3 Suppl1):43-48
`
`[1997]). However, despite the association ofErbB2 overexpression with poor prognosis, the
`
`odds ofHER2-positive patients responding clinically to treatment with taxanes were greater than
`
`three times those ofHER2-negative patients (Ibid).
`rhuMab HER2 was shown to enhance the
`activity of paclitaxel (T AXOL ®) and doxorubicin against breast cancer xenografts in nude mice
`
`10
`
`injected withBT-474 human breast adenocarcinoma cells, which express high levels ofHER2
`
`(Baselga et al., Breast Cancer, Proceedings of ASCO, Vol. 13, Abstract 53 [1994]).
`
`Summary of the Invention
`
`The present invention concerns the treatment of disorders characterized by overexpression
`
`ofErbB2, and is based on the recognition that while treatment with anti-ErbB2 antibodies
`
`markedly enhances the clinical benefit of the use of chemotherapeutic agents in general, a
`
`syndrome of myocardial dysfunction that has been observed as a side-effect of anthracycline
`
`derivatives is increased by the administration of anti-ErbB2 antibodies.
`
`Accordingly, the invention concerns a method for the treatment of a human patient
`
`susceptible to or diagnosed with a disorder characterized by overexpression ofErbB2 receptor
`
`2~
`
`·::.":;t,'i'
`
`comprising administering a therapeutically effective amount of a combination of an anti-ErbB2
`
`antibody and a chemotherapeutic agent other than an anthracycline derivative, e.g. doxorubicin or
`
`epirubicin, in the absence of an anthracycline derivative, to the human patient.
`
`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
`
`25
`
`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, vulval cancer, thyroid cancer, hepatic carcinoma and various types
`
`of head and neck cancer. The chemotherapeutic agent preferably is a taxoid, such as T AXOL ®
`
`5
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`HOSPIRA EX. 1121
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`(paclitaxel) or a T AXOL ® derivative.
`
`Although an antiproliferative effect is sufficient, in a preferred embodiment, the anti(cid:173)
`
`ErbB2 antibody is capable of inducing cell death or is capable of inducing apoptosis. Preferred
`
`anti-ErbB2 antibodies bind the extracellular domain of the ErbB2 receptor, and preferably bind to
`
`5
`
`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.
`
`The method of the present invention is particularly suitable for the treatment of breast or
`
`ovarian cancer, characterized by the overexpression of the ErbB2 receptor.
`
`In another aspect, the invention concerns an article of manufacture, comprising a
`
`10
`
`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 characterized by overexpression ofErbB2 receptor, and a package insert
`
`containing instructions to avoid the use of anthracycline-type chemotherapeutics in combination
`
`with the composition.
`
`Brief Description of the Drawings
`
`Fig. 1 shows epitope-mapping of the extracellular domain ofErbB2 as determined by
`
`truncation mutant analysis and site-directed mutagenesis (Nakamura et al. J of Virology
`
`67(10):6179-6191 [Oct 1993]; Renz et al. J. Cell Bioi. 125(6): 1395-1406 [Jun 1994]). The anti(cid:173)
`
`proliferative MAbs 4D5 and 3H4 bind adjacent to the transmembrane domain. The various
`
`26
`
`ErbB2-ECD truncations or point mutations were prepared from eDNA using polymerase chain
`
`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
`
`SV 40 termination and polyadenylation signals located downstream of the inserted eDNA.
`
`Plasmid DNA was transfected into 293 S cells. One day following transfection, the cells were
`
`25
`
`metabolically labeled overnight in methionine and cysteine-free, low glucose DMEM containing
`1% dialyzed fetal bovine serum and 25 )JCi each of 35S methionine and 35S cysteine. Supernatants
`
`were harvested 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
`
`6
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`HOSPIRA EX. 1121
`Page 10
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`

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`5
`
`10
`
`analyzed by 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 ofErbB2 (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).
`Detailed Description of the Preferred Embodiments
`I.
`Definitions
`
`The terms "HER2", "ErbB2" "c-Erb-B2" are used interchangeably. Unless indicated
`
`otherwise, the terms "ErbB2" "c-Erb-B2" and "HER2" when used herein refer to the human
`protein and "her2", "erbB2" and "c-erb-B2" refer to human gene. The human erbB2 gene and
`
`ErbB2 protein are, for example, described in Semba et al., PNAS (USA) 82:6497-6501 (1985) and
`
`Yamamoto et al. Nature 319:230-234 (1986) (Genebank accession number X03363). ErbB2
`
`comprises four domains (Domains 1-4).
`
`The "epitope 4D5" is the region in the extracellular domain ofErbB2 to which the
`
`antibody 4D5 (ATCC CRL 10463) binds. This epitope is close to the transmembrane region of
`
`ErbB2. To screen for antibodies which bind to the 4D5 epitope, a routine cross-blocking assay
`
`such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed
`
`Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be
`
`performed (see Fig. 1) to assess whether the antibody binds to the 4D5 epitope ofErbB2 (i.e. any
`one or more residues in the region from about residue 529, e.g. about residue 561 to about
`
`residue 625, inclusive).
`
`The "epitope 3H4" is the region in the extracellular domain ofErbB2 to which the
`
`antibody 3H4 binds. This epitope is shown in Fig. 1, and includes residues from about 541 to
`
`about 599, inclusive, in the amino acid sequence ofErbB2 extracellular domain.
`
`25
`
`The "epitope 7C2/7F3" is the region at theN terminus of the extracellular domain of
`
`ErbB2 to which the 7C2 and/or 7F3 antibodies (each deposited with the ATCC, see below) bind.
`
`To screen for antibodies which bind to the 7C2/7F3 epitope, a routine cross-blocking assay such
`
`as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed
`
`Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping can be
`
`7
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`performed to establish whether the antibody binds to the 7C2/7F3 epitope on ErbB2 (i.e. any one
`
`or more of residues in the region from about residue 22 to about residue 53 ofErbB2; SEQ ID
`
`N0:2).
`
`The term "induces cell death" or "capable of inducing cell death" refers to the ability of
`
`5
`
`the antibody to make a viable cell become nonviable. The "cell" here is one which expresses the
`
`ErbB2 receptor, especially where the cell overexpresses the ErbB2 receptor. A cell which
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`"overexpresses" ErbB2 has significantly higher than normal ErbB2 levels compared to a
`
`noncancerous cell of the same tissue type. Preferably, the cell is a cancer cell, e.g. a breast,
`
`ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder
`
`10
`
`cell. In vitro, the cell may be a SKBR3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or
`
`SKOV3 celL Cell death in vitro may be determined in the absence of complement and immune
`
`effector cells to distinguish cell death induced by antibody dependent cellular cytotoxicity
`
`(ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be
`
`performed using heat inactivated serum (i.e. in the absence of complement) and in the absence of
`
`immune effector cells. To determine whether the antibody is able to induce cell death, loss of
`
`membrane integrity as evaluated by uptake ofpropidium iodide (PI), trypan blue (see Moore et al.
`Cytotechnology 17: 1-11 [ 1995]) or 7 AAD can be assessed relative to untreated cells. Preferred
`
`cell death-inducing antibodies are those which induce PI uptake in the "PI uptake assay in BT474
`
`cells".
`
`2@':1
`
`The phrase "induces apoptosis" or "capable of inducing apoptosis" refers to the ability of
`
`the antibody to induce programmed cell death as determined by binding of annexin V,
`
`fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,
`
`and/or formation of membrane vesicles (called apoptotic bodies). The cell is one which
`
`overexpresses the ErbB2 receptor. Preferably the "cell" is a tumor cell, e.g. a breast, ovarian,
`
`25
`
`stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. In
`
`vitro, the cell may be a SKBR3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or SKOV3
`
`cell. Various methods are available for evaluating the cellular events associated with apoptosis.
`
`For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA
`
`fragmentation can be evaluated through DNA laddering as disclosed in the example herein; and
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`nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase
`
`in hypodiploid cells. Preferably, the antibody which induces apoptosis is one which results in
`
`about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold,
`
`induction ofannexin binding relative to untreated cell in an "annexin binding assay using BT474
`
`5
`
`cells" (see below).
`
`Sometimes the pro-apoptotic antibody will be one which blocks HRG binding/activation
`
`of the ErbB2/ErbB3 complex (e.g. 7F3 antibody). In other situations, the antibody is one which
`
`does not significantly block activation ofthe ErbB2/ErbB3 receptor complex by HRG (e.g. 7C2).
`
`Further, the antibody may be one like 7C2 which, while inducing apoptosis, does not induce a
`
`10
`
`large reduction in the percent of cells in S phase (e.g. one which only induces about 0-10%
`
`reduction in the percent of these cells relative to control).
`
`The antibody of interest may be one like 7C2 which binds specifically to human ErbB2 and
`
`does not significantly cross-react with other proteins such as those encoded by the erbB 1, erbB3
`
`and/or erbB4 genes. Sometimes, the antibody may not significantly cross-react with the rat neu
`
`protein, e.g., as described in Schecter et al. Nature 312:513 (1984) and Drebin et al., Nature
`
`312:545-548 {1984). In such embodiments, the extent of binding of the antibody to these
`
`proteins (e.g., cell surface binding to endogenous receptor) will be less than about 10% as
`
`determined by fluorescence activated cell sorting (F ACS) analysis or radioimmunoprecipitation
`
`(RIA).
`
`2~
`
`"Heregulin" (HRG) when used herein refers to a polypeptide which activates the ErbB2-
`
`ErbB3 and ErbB2-ErbB4 protein complexes (i.e. induces phosphorylation of tyrosine residues In
`
`the complex upon binding thereto). Various heregulin polypeptides encompassed by this term are
`
`disclosed in Holmes et al., Science, 256:1205-1210 (1992); WO 92/20798; Wen et al., Mol. Cell.
`
`Bioi., 14(3):1909-1919 (1994); and Marchionni et al., Nature, 362:312-318 (1993), for example.
`
`25
`
`The term includes biologically active fragments and/or variants of a naturally occurring HRG
`
`polypeptide, such as an EGF-like domain fragment thereof (e.g. HRGP 1177•244 ).
`The "ErbB2-ErbB3 protein complex" and "ErbB2-ErbB4 protein complex" are
`
`noncovalently associated oligomers of the ErbB2 receptor and the ErbB3 receptor or ErbB4
`
`receptor, respectively. The complexes form when a cell expressing both of these receptors is
`
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`exposed to HRG and can be isolated by immunoprecipitation and analyzed by SDS-P AGE as
`
`described in Sliwkowski eta!., J. Bioi. Chern., 269(20):14661-14665 (1994).
`
`"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same
`
`structural characteristics. While antibodies exhibit binding specificity to a specific antigen,
`
`5
`
`immunoglobulins include both antibodies and other antibody-like molecules which lack antigen
`
`specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph
`
`system and at increased levels by myelomas.
`
`"Native antibodies" and "native immunoglobulins" are usually heterotetrameric
`
`glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two
`
`10
`
`identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide
`
`bond, while the number of disulfide linkages varies among the heavy chains of different
`
`immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain
`disulfide bridges. Each heavy chain has at one end a variable domain (V tJ followed by a number
`
`of constant domains. Each light chain has a variable domain at one end (V1) and a constant
`domain at its other end; the constant domain of the light chain is aligned with the first constant
`
`domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain
`
`of the heavy chain. Particular amino acid residues are believed to form an interface between the
`
`light- and heavy-chain variable domains.
`
`The term "variable" refers to the fact that certain portions of the variable domains differ
`
`2~
`
`~=F
`
`extensively in sequence among antibodies and are used in the binding and specificity of each
`
`particular antibody for its particular antigen. However, the variability is not evenly distributed
`
`throughout the variable domains of antibodies. It is concentrated in three segments called
`
`complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and
`
`the heavy-chain variable domains. The more highly conserved portions of variable domains are
`
`25
`
`called the framework region (FR). The variable domains of native heavy and light chains each
`
`comprise four FR regions, largely adopting a P-sheet configuration, connected by three CDRs,
`
`which form loops connecting, and in some cases forming part of, the P-sheet structure. The
`
`CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the
`
`other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat eta!.,
`
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`NIH Pub/. No.91-3242, Vol. I, pages 647-669 [1991]). The constant domains are not involved
`
`directly in binding an antibody to an antigen, but exhibit various effector functions, such as
`
`participation of the antibody in antibody dependent cellular cytotoxicity.
`
`Papain digestion of antibodies produces two identical antigen-binding fragments, called
`
`5
`
`"Fab" fragments, each with a single antigen-binding site, and a residual "Fe" fragment, whose
`
`name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has
`
`two antigen-combining sites and is still capable of cross-linking antigen.
`
`"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and
`
`-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in
`
`10
`
`tight, non-covalent association. It is in this configuration that the three CDRs of each variable
`
`domain interact to define an antigen-binding site on the surface of the V H-V L dimer. Collectively,
`
`the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable
`
`domain (or hal