`
`
`
`
`
`
`PHIGENIX
`PHIGENIX
`Exhibit 1025
`Exhibit 1025
`
`

`

`(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2003/0170235 A1
`
`
` Cohen (43) Pub. Date: Sep. 11, 2003
`
`US 20030170235A1
`
`(54) TREATMENT WITH ANTI-ERBBZ
`ANTIBODIES
`
`Related US, Application Data
`
`(75)
`
`Inventor: Robert L. Cohen, San Mateo, CA (US)
`
`Correspondence Address:
`GENENTECH, INC.
`1 DNA WAY
`SOUTH SAN FRANCISCO, CA 94030 (US)
`
`(73) Assignee: Genentech, Inc.
`
`(21) Appl. N0.:
`
`10/429,519
`
`(22)
`
`Filed:
`
`May 5, 2003
`
`(63) Continuation of application No. 09/568,322, filed on
`May 9, 2000.
`
`(60) Provisional application No. 60/134,085, filed on May
`14, 1999.
`
`Publication Classification
`
`Int. Cl.7 ...................... A61K 39/395; A61K 31/337
`(51)
`(52) US. Cl.
`......................................... 424/143.1; 514/449
`
`(57)
`
`ABSTRACT
`
`The present invention concerns the treatment of cancer with
`anti-ErbB2 antibodies.
`
`PHIGENIX
`
`Exhibit 1025-01
`
`

`

`Patent Application Publication
`
`Sep. 11, 2003
`
`US 2003/0170235 A1
`
`3H4
`405
`702
`7F3
`
`300
`
`aa 541—599
`aa 529—625
`aa 22—53
`aa 22—53
`
`400
`
`1 00
`
`‘
`
`200
`
`
`
`3H4 epitope (SEQ ID NO:3)
`
`VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVAR
`|
`.
`|
`541
`599
`
`A
`
`.
`
`4D5 epitope (SEQ ID NO:4)
`
`LPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQP
`I
`|
`561
`625
`14
`FIG... 1
`
`1
`
`38
`
`75
`
`MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPA
`
`SPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFL
`
`QDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDN
`
`112 YALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEI
`
`149
`186
`
`LKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLID
`TNRSRA
`
`FIG._2
`
`PHIGENIX
`
`Exhibit 1025-02
`
`

`

`US 2003/0170235 A1
`
`Sep. 11, 2003
`
`TREATMENT WITH ANTI-ERBB2 ANTIBODIES
`
`RELATED APPLICATIONS
`
`[0001] This application is a continuation application of
`non-provisional application Ser. No. 09/568,322 filed May
`9, 2000 under 37 CFR 1.53(b)(1), claiming priority under 35
`USC 119(e) to provisional application No. 60/134,085 filed
`May 14, 1999, the contents of which are incorporated herein
`by reference.
`
`FIELD OF THE INVENTION
`
`[0002] The present invention concerns the treatment of
`cancer with anti-ErbB2 antibodies.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Proto-oncogenes that encode growth factors and
`growth factor receptors have been identified to play impor-
`tant roles in the pathogenesis of various human malignan-
`cies,
`including breast cancer. It has been found that the
`human erbB2 gene (also known as HER2, or c-erbB-2),
`which encodes a 185 -kd transmembrane glycoprotein recep-
`tor (p185HER2) related to the epidermal growth factor recep-
`tor (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]).
`
`[0004] 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
`237:178-182 [1987]). Transgenic mice that express HER2
`were found to develop mammary tumors (Guy et al., Proc.
`Natl. Acad. Sci. USA 89:10578-10582 [1992]).
`
`[0005] Antibodies directed against human ErbB2 protein
`and the protein 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 surface p185
`expression on B104-1-1 cells (NIH-3T3 cells transfected
`with the neu protooncogene) and inhibits colony formation
`of these cells. In Drebin et al., PNAS (USA) 83:9129-9133
`(1986), the 7.16.4 antibody was shown to inhibit the tum-
`origenic 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 production of
`a panel of antibodies against the rat neu gene product. All of
`the antibodies were found to exert a cytostatic effect on the
`growth of neu-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 anti-
`bodies were able to mediate high levels of antibody depen-
`dent 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 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
`WO94/22478 published Oct. 13, 1994.
`
`[0006] 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 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. 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 sensi-
`tize p185erbB2-overexpressing breast tumor cell lines to the
`cytotoxic effects of TNF-ot. See also WO89/06692 pub-
`lished 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 (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 921829-1838 (1994); Vitetta
`et al. 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).
`
`[0007] Tagliabue et al. Int. J. Cancer 47:933-937 (1991)
`describe two antibodies which were selected for their reac-
`
`tivity 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.
`
`[0008] McKenzie et al. Oncogene 4:543-548 (1989) gen-
`erated a panel of anti-ErbB2 antibodies with varying epitope
`specificities, including the antibody designated TA1. This
`TA1 antibody was found to induce accelerated endocytosis
`of ErbB2 (see Maier et al. Cancer Res. 51:5361-5369
`(1991)). Bacus et al. Molecular Carcinogenesis 3:350-362
`(1990) reported that the TA1 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 of ErbB2
`
`receptor at the cell surface and transient increased levels in
`the cytoplasm.
`
`al. PNAS (USA) 88:8691-8695
`[0009] Stancovski et
`(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 tumor growth. Monoclonal
`antibody N28 induced significant phosphorylation of the
`ErbB2 receptor, whereas the other four antibodies generally
`displayed low or no phosphorylation-inducing activity. The
`effect of the anti-ErbB2 antibodies on proliferation of
`SKBR3 cells was also assessed. In this SKBR3 cell prolif-
`
`PHIGENIX
`
`Exhibit 1025-03
`
`

`

`US 2003/0170235 A1
`
`Sep. 11, 2003
`
`eration assay, two of the antibodies (N12 and N29) caused
`a reduction in cell proliferation relative to control. The
`ability of the various antibodies to induce cell lysis in vitro
`via complement-dependent cytotoxicity (CDC) and anti-
`body dependent cellular 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.
`
`rhuMab HER2 was shown to enhance the activity of pacli-
`taxel (TAXOL®) and doxorubicin against breast cancer
`xenografts in nude mice injected with BT-474 human breast
`adenocarcinoma cells, which express high levels of HER2
`(Baselga et al., Breast Cancer; Proceedings ofASCO, Vol.
`13, Abstract 53 [1994]).
`
`SUMMARY OF THE INVENTION
`
`[0010] Bacus et al. Cancer Research 52:2580-2589 (1992)
`further characterized the antibodies 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 iv. 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 inhibi-
`tion was also observed with the N24 antibody. Bacus et al.
`also tested the ability of the antibodies to promote a mature
`phenotype in the human breast cancer cell lines AU-5 65 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.
`
`[0011] Xu et al. Int. J. Cancer 53:401-408 (1993) evalu-
`ated a panel of anti-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 combina-
`tions), modulate cell-surface ErbB2, and inhibit
`ligand
`stimulated anchorage-independent growth. See also WO94/
`00136 published Jan. 6, 1994 and Kasprzyk et al. Cancer
`Research 52:2771-2776 (1992) concerning anti-ErbB2 anti-
`body combinations. Other anti-ErbB2 antibodies are dis-
`cussed in Hancock et al. 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.
`Biol. Chem. 267:15160-15167 (1992).
`
`[0012] A recombinant humanized anti-ErbB2 monoclonal
`antibody (a humanized version of the murine anti-ErbB2
`antibody 4D5, referred to as rhuMAb HER2 or HERCEP-
`TIN®, has been clinically active in patients with ErbB2-
`overexpressing metastatic breast cancers that had received
`extensive prior anticancer therapy. (Baselga et al., J. Clin.
`Oncol. 14:737-744 [1996]).
`
`[0013] ErbB2 overexpression is commonly regarded as a
`predictor of a poor prognosis, especially in patients with
`primary disease that involves axillary lymph nodes (Slamon
`et al., [1987] and [1989], supra; Ravdin and Chamness,
`Gene 159:19-27 [1995]; and Hynes and Stern, Biochim
`BiophysActa 1198:165-184 [1994]), and has been linked to
`sensitivity and/or resistance to hormone therapy and che-
`motherapeutic regimens,
`including CMF (cyclophospha-
`mide, methotrexate, and fiuoruracil) and anthracyclines
`(Baselga et al., Oncology 11(3 Suppl 2):43-48 [1997]).
`However, despite the association of ErbB2 overexpression
`with poor prognosis, the odds of HER2-positive patients
`responding clinically to treatment with taxanes were greater
`than three times those of HER2-negative patients (Ibid).
`
`In a first aspect, the present invention provides a
`[0014]
`method of treating a human patient susceptible to or diag-
`nosed with a tumor in which ErbB2 protein is expressed
`comprising the following steps, performed sequentially:
`
`(a) treating the patient with a therapeutically
`[0015]
`effective amount of an anti-ErbB2 antibody and,
`optionally, further comprising treating the patient
`with a therapeutically effective amount of a chemo-
`therapeutic agent (e.g. a taxoid, such as paclitaxel or
`doxetaxel);
`
`[0016]
`
`(b) surgically removing the tumor; and then
`
`(c) treating the patient with a therapeutically
`[0017]
`effective amount of an anti-ErbB2 antibody and/or of
`a chemotherapeutic agent (e.g. a taxoid, such as
`paclitaxel or doxetaxel).
`
`[0018] Preferably, the tumor overexpresses ErbB2 protein
`and is selected from the group consisting of a breast tumor,
`squamous cell tumor, small-cell lung tumor, non-small cell
`lung tumor, gastrointestinal tumor, pancreatic tumor, glio-
`blastoma, cervical tumor, ovarian tumor, liver tumor, blad-
`der
`tumor, hepatoma, colon tumor, colorectal
`tumor,
`endometrial
`tumor, salivary gland tumor, kidney tumor,
`prostate tumor, vulval tumor, thyroid tumor, hepatic tumor,
`head tumor and neck tumor.
`
`[0019] The invention further provides an article of manu-
`facture comprising a container, a composition within the
`container comprising an anti-ErbB2 antibody and a package
`insert instructing the user of the composition to treat a
`patient essentially according to the above method.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0020] FIG. 1 shows epitope-mapping of the extracellular
`domain of ErbB2 as determined by truncation mutant analy-
`sis and site-directed mutagenesis (Nakamura et al. J. of
`Virology 67(10):6179-6191 [October 1993]; Renz et al. J.
`Cell Biol. 125(6):1395-1406 [June 1994]). The anti-prolif-
`erative MAbs 4D5 and 3H4 bind adjacent to the transmem-
`brane domain. The various ErbB2-ECD truncations or point
`mutations were prepared from cDNA 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 SV40 termination and polyadeny-
`lation signals located downstream of the inserted cDNA.
`Plasmid DNA was transfected into 2938 cells. One day
`following transfection, the cells were metabolically labeled
`overnight
`in methionine and cysteine-free,
`low glucose
`DMEM containing 1% dialyzed fetal bovine serum and 25
`nCi each of 35$ methionine and 35$ 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 electrophoresed at 100 V. The gel
`
`PHIGENIX
`
`Exhibit 1025-04
`
`

`

`US 2003/0170235 A1
`
`Sep. 11, 2003
`
`was electroblotted onto a membrane and analyzed by auto-
`radiography. SEQ ID NOs23 and 4 depict the 3H4 and 4D5
`epitopes, respectively.
`
`[0021] FIG. 2 depicts with underlining the amino acid
`sequence of Domain 1 of ErbB2 (SEQ ID NO21). 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 NO22).
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`
`[0022]
`
`I. Definitions
`
`[0023] The terms “HER2”, “ErbB2” and “c-Erb-B2” are
`used interchangeably. Unless indicated otherwise, the terms
`“ErbB2”, “c-Erb-B2” and “HER2” refer to the human pro-
`tein, 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) 8226497-
`6501 (1985) and Yamamoto et al. Nature 3192230-234
`(1986) (Genebank accession number X03363). ErbB2 com-
`prises four domains (Domains 1-4).
`
`[0024] The “epitope 4D5” is the region in the extracellular
`domain of ErbB2 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 per-
`formed (see FIG. 1) to assess whether the antibody binds to
`the 4D5 epitope of ErbB2 (i.e. any one or more residues in
`the region from about residue 529, e.g. about residue 561 to
`about residue 625, inclusive; SEQ ID NO24).
`
`[0025] The “epitope 3H4” is the region in the extracellular
`domain of ErbB2 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 of
`ErbB2 extracellular domain (SEQ ID NO23).
`
`the N
`[0026] The “epitope 7C2/7F3” is the region at
`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 per-
`formed 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 of
`ErbB2 [SEQ ID NO:2]).
`
`[0027] The term “induces cell death” or “capable of induc-
`ing cell death” refers to the ability of 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 “overex-
`presses” 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 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 deter-
`mined 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 of
`propidium iodide (PI), trypan blue (see Moore et al. Cyto-
`technology 1721-11 [1995]) or 7AAD can be assessed rela-
`tive to untreated cells. Preferred cell death-inducing anti-
`bodies are those which induce PI uptake in the “PI uptake
`assay in BT474 cells”.
`
`[0028] 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
`endoplasmatic reticulum, cell fragmentation, and/or forma-
`tion 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, 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 nuclear/chromatin condensation along with DNA frag-
`mentation 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
`of annexin binding relative to untreated cell in an “annexin
`binding assay using BT474 cells” (see below).
`
`[0029] 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 anti-
`body is one which does not significantly block activation of
`the 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 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).
`
`[0030] 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 erbB1, erbB3 and/or erbB4 genes. Some-
`times, the antibody may not significantly cross-react with
`the rat neu protein, e.g., as described in Schecter et al.
`Nature 3122513 (1984) and Drebin et al., Nature 3122545-
`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 deter-
`mined by fluorescence activated cell sorting (FACS) analy-
`sis or radioimmunoprecipitation (RIA).
`
`“Heregulin” (HRG) when used herein refers to a
`[0031]
`polypeptide which activates the ErbB2-ErbB3 and ErbB2-
`ErbB4 protein complexes (i.e. induces phosphorylation of
`
`PHIGENIX
`
`Exhibit 1025-05
`
`

`

`US 2003/0170235 A1
`
`Sep. 11, 2003
`
`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. Biol,
`14(3):1909-1919 (1994); and Marchionni et al., Nature,
`362:312-318 (1993), for example. The term includes bio-
`logically active fragments and/or variants of a naturally
`occurring HRG polypeptide, such as an EGF-like domain
`fragment thereof (e.g. HRGB1177_244).
`
`[0032] The “ErbB2-ErbB3 protein complex” and “ErbB2-
`ErbB4 protein complex” are noncovalently associated oli-
`gomers of the ErbB2 receptor and the ErbB3 receptor or
`ErbB4 receptor, respectively. The complexes form when a
`cell expressing both of these receptors is exposed to HRG
`and can be isolated by immunoprecipitation and analyzed by
`SDS-PAGE as described in Sliwkowski et al., J. Biol.
`Chem, 269(20):14661-14665 (1994).
`
`“Antibodies” (Abs) and “immunoglobulins” (Igs)
`[0033]
`are glycoproteins having the same structural characteristics.
`While antibodies exhibit binding specificity to a specific
`antigen, 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”
`[0034]
`are usually heterotetrameric glycoproteins of about 150,000
`daltons, composed of two identical light (L) chains and two
`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 (VH)
`followed by a number of constant domains. Each light chain
`has a variable domain at one end (VL) 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.
`
`[0035] The term “variable” refers to the fact that certain
`portions of the variable domains differ 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 concen-
`trated 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 called the
`framework region (FR). The variable domains of native
`heavy and light chains each comprise four FR regions,
`largely adopting B-sheet configuration, connected by three
`CDRs, which form loops connecting, and in some cases
`forming part of, the B-sheet structure. The CDRs in each
`chain are held together in close proximity by the FR regions
`and, with the CDRs from the other chain, contribute to the
`formation of the antigen-binding site of antibodies (see
`Kabat et al., NIH Publ. No.91-3242, Vol. 1, pages 647-669
`[1991]). The constant domain not involved directly in bind-
`
`ing an antibody to an antigen, but exhibit various effector
`functions, such as participation of the antibody in antibody-
`dependent cellular toxicity.
`
`[0036] Papain digestion of antibodies produces two iden-
`tical antigen-binding fragments, called “Fab” fragments,
`each with a single antigen-binding site, and a residual “Fc”
`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
`[0037]
`contains a complete antigen-recognition and -binding site.
`This region consists of a dimer of one heavy- and one
`light-chain variable domain in tight, non-covalent associa-
`tion. 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 VH-VL dimer. Collectively, the six CDRs
`confer antigen-binding specificity to the antibody. However,
`even a single variable domain (or half of an Fv comprising
`only three CDRs specific for an antigen) has the ability to
`recognize and bind antigen, although at a lower affinity than
`the entire binding site.
`
`[0038] The Fab fragment also contains the constant
`domain of the light chain and the first constant domain
`(CH1) of the heavy chain. Fab fragments differ from Fab
`fragments by the addition of a few residues at the carboxy
`terminus of the heavy chain CH1 domain including one or
`more cysteines, from the antibody hinge region. Fab'-SH is
`the designation herein for Fab' in which the cysteine resi-
`due(s) of the constant domains bear a free thiol group.
`F(ab')2 antibody fragments originally were produced as pairs
`of Fab' fragments which have hinge cysteines between them.
`Other chemical couplings of antibody fragments are also
`known.
`
`[0039] The “light chains” of antibodies (immunoglobu-
`lins) from any vertebrate species can be assigned to one of
`two clearly distinct types, called kappa (K) and lambda ()t),
`based on the amino acid sequences of their constant
`domains.
`
`[0040] Depending on the amino acid sequence of the
`constant domain of their heavy chains, immunoglobulins
`can be assigned to different classes. There are five major
`classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM,
`and several of these may be further divided into subclasses
`(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The
`heavy-chain constant domains that correspond to the differ-
`ent classes of immunoglobulins are called (X, 5, e, y, and M,
`respectively. The subunit structures and three-dimensional
`configurations of different classes of immunoglobulins are
`well known.
`
`[0041] The term “antibody” is used in the broadest sense
`and specifically covers intact monoclonal antibodies, poly-
`clonal antibodies, multispecific antibodies (e.g. bispecific
`antibodies) formed from at least two intact antibodies, and
`antibody fragments so long as they exhibit
`the desired
`biological activity.
`
`“Antibody fragments” comprise a portion of an
`[0042]
`intact antibody, preferably the antigen binding or variable
`region of the intact antibody. Examples of antibody frag-
`ments include Fab, Fab', F(ab')2, and Fv fragments; diabod-
`ies; linear antibodies (Zapata et al. Protein Eng. 8(10):1057-
`
`PHIGENIX
`
`Exhibit 1025-06
`
`

`

`US 2003/0170235 A1
`
`Sep. 11, 2003
`
`and
`single-chain antibody molecules;
`[1995]);
`1062
`multispecific antibodies formed from antibody fragments.
`
`[0043] The term “monoclonal antibody” as used herein
`refers to an antibody obtained from a population of substan-
`tially homogeneous antibodies, i. e., the individual antibod-
`ies comprising the population are identical except for pos-
`sible naturally occurring mutations that may be present in
`minor amounts. Monoclonal antibodies are highly specific,
`being directed against a single antigenic site. Furthermore,
`in contrast to conventional (polyclonal) antibody prepara-
`tions which typically include different antibodies directed
`against different determinants (epitopes), each monoclonal
`antibody is directed against a single determinant on the
`antigen.
`In addition to their specificity,
`the monoclonal
`antibodies are advantageous in that they are synthesized by
`the hybridoma culture, uncontaminated by other immuno-
`globulins. The modifier “monoclonal” indicates the charac-
`ter of the antibody as being obtained from a substantially
`homogeneous population of antibodies, and is not to be
`construed as requiring production of the antibody by any
`particular method. For example, the monoclonal antibodies
`to be used in accordance with the present invention may be
`made by the hybridoma method first described by Kohler et
`al., Nature, 256:495 (1975), or may be made by recombinant
`DNA methods (see, e.g., US. Pat. No.4,816,567). The
`“monoclonal antibodies” may also be isolated from phage
`antibody libraries using the techniques described in Clack-
`son et al., Nature, 352:624-628 (1991) and Marks et al., J.
`Mol. Biol, 222:581-597 (1991), for example.
`
`[0044] The monoclonal antibodies herein specifically
`include “chimeric” antibodies (immunoglobulins) in which
`a portion of the heavy and/or light chain is identical with or
`homologous to corresponding sequences in antibodies
`derived from a particular species or belonging to a particular
`antibody class or subclass, while the remainder of the
`chain(s) is identical with or homologous to corresponding
`sequences in antibodies derived from another species or
`belonging to another antibody class or subclass, as well as
`fragments of such antibodies, so long as they exhibit the
`desired biological activity (US. Pat. No. 4,816,567; Morri-
`son et al.,Proc. Natl.Acaa'. Sci. USA, 81:6851-6855 (1984)).
`
`“Humanized” forms of non-human (e.g., murine)
`[0045]
`antibodies are chimeric immunoglobulins, immunoglobulin
`chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or
`other antigen-binding subsequences of antibodies) which
`contain minimal sequence derived from non-human immu-
`noglobulin. For the most part, humanized antibodies are
`human immunoglobulins (recipient antibody) in which resi-
`dues from a complementarity-determining region (CDR) of
`the recipient are replaced by residues from a CDR of a
`non-human species (donor antibody) such as mouse, rat or
`rabbit having the desired specificity, affinity, and capacity. In
`some instances, Fv framework region (FR) residues of the
`human immunoglobulin are replaced by corresponding non-
`human residues. Furthermore, humanized antibodies may
`comprise residues which are found neither in the recipient
`antibody nor in the imported CDR or framework sequences.
`These modifications are made to further refine and maximize
`
`antibody performance. In general, the humanized antibody
`will comprise substantially all of at least one, and typically
`two, variable domains, in which all or substantially all of the
`CDR regions correspond to those of a non-human immu-
`noglobulin and all or substantially all of the FR regions are
`
`those of a human immunoglobulin sequence. The human-
`ized antibody optimally also will comprise at least a portion
`of an immunoglobulin constant region (Fc), typically that of
`a human immunoglobulin. For further details, see Jones et
`al., Nature, 321:522-525 (1986); Reichmann et al., Nature,
`332:323-329 (1988); and Presta, Curr. 0p. Struct. Biol,
`2:593-596 (1992). The humanized antibody includes a PRI-
`MATIZEDTM antibody wherein the antigen-binding region
`of the antibody is derived from an antibody produced by
`immunizing macaque monkeys with the antigen