`Patentamt
`European
`Patent Office
`Office europeen
`des brevets
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`EP 1 037 926 81
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`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`15.01.2014 Bulletin 2014/03
`
`(21) Application number: 98963840.8
`
`(22) Date of filing: 10.12.1998
`
`(51) lnt Cl.:
`A61K 38/00(2006.01J
`A61 K 391395 (2006.01J
`A61K 3117068(2006.01)
`
`C07K 16132 (2006·01J
`A61K 45106 (2006·01)
`
`(86) International application number:
`PCT/US1998/026266
`
`(87) International publication number:
`WO 1999/031140 (24.06.1999 Gazette 1999/25)
`
`(54) TREATMENT WITH ANTI-ErbB2 ANTIBODIES
`
`BEHANDLUNG MIT ANTIKORPERN GEGEN ERBB2
`
`TRAITEMENT AUX ANTICORPS ANTI-ErbB2
`
`(84) Designated Contracting States:
`AT BE CH CY DE DK ES Fl FR GB GR IE IT Ll LU
`MC NL PT SE
`Designated Extension States:
`AL LT LV MK RO Sl
`
`(74) Representative: Cripps, Joanna Elizabeth et al
`Mewburn Ellis LLP
`33 Gutter Lane
`London
`EC2V BAS (GB)
`
`(30) Priority: 12.12.1997 US 69346 P
`
`(43) Date of publication of application:
`27.09.2000 Bulletin 2000/39
`
`(60) Divisional application:
`08002425.0 /1 947 119
`10177992.4/2 275 450
`10178009.6/2 277 919
`
`(73) Proprietor: Genentech, Inc.
`South San Francisco CA 94080-4990 (US)
`
`(72) Inventors:
`• SHAK, Steven
`Burlingame, CA 94010 (US)
`• PATON, Virginia, E.
`Oakland, CA 94606 (US)
`• DESMOND-HELLMANN, Susan Diane of
`Genentech, inc.
`CA-94080 South San Francisco (US)
`
`(56) References cited:
`WO-A-97/20858
`US-A- 5 705 157
`
`US-A- 5 677 171
`
`• BASELGA J. ET AL.: "HER2 Overexpression and
`Paclitaxel sensitivity in breast cancer:
`Therapeutic implications" ONCOLOGY, vol. 11,
`no. 3, March 1997, pages 43-48, XP002100077
`• MENDELSOHN J. ET AL.: "Receptor blockade
`and chemotherapy: a new approach to
`combination cancer therapy" ANNALS OF
`ONCOLOGY, vol.7, suppl.1, 1996, page 22
`XP0021 00078
`• BASELGA J. ET AL.:"Monoclonal antibodies
`directed against growth factor receptors enhance
`the efficacy of chemotherapeutic agents"
`ANNALS OF ONCOLOGY, vo1.5, supp1.5, 1994,
`page A010 XP002100164
`
`Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent
`Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the
`Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been
`paid. (Art. 99(1) European Patent Convention).
`
`c..
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`Description
`
`Field of the Invention
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`[0001] The present invention concerns the treatment of disorders characterized by the overexpression of with malignant
`breast Erb82. More specifically, the invention concerns the treatment of human patients with malignant breast cancer
`overexpressing ErbB2 with a combination of an anti-ErbB2 antibody and a chemotherapeutic agent that is a taxoid, in
`the absence of an anthracycline, e.g. doxorubicin or epirubicin.
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`Background of the Invention
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`[0002] 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 breast cancer. It has been found that the human
`Erb82 gene (erb82, 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 (Siamon et al., Science 235:177-182 [1987]; Slamon et al., Science 244:707-712 [1989]).
`[0003] Several lines of evidence support a direct role for ErbB2 in the pathogenesis and clinical aggressiveness of
`Erb82-overexpressing tumors. The introduction of Erb82 into non-neoplastic cells has been shown to cause their ma(cid:173)
`lignant 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 develop mammary tumors (Guy et al. Proc. Natl.
`Acad. Sci. USA 89:10578-1 0582 [1992]).
`[0004] Antibodies directed against human erb82 protein products and proteins encoded by the rat equivalent of the
`erb82 gene (neu) have been described. Drebin et al., Cell41:695-706 (1985) refer to an lgG2a 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 8104-1-1 cells (NIH-3T3 cells transfected with the neu proto-oncogene) and inhibits colony formation
`of these cells. In Drebin et al. PNAS (USA) 83:9.129-9133 (1986), the 7.16.4 antibody was shown to 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 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 lgM, lgG2a and lgG2b 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 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.
`[0005] 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%.
`Other antibodies in the panel, including 7C2 and 7F3, reduced cellular proliferation to a lesser extent in this assay.
`Hudziak eta/. 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 p 185erb82-overexpressing breast tumor cell lines to the cytotoxic effects of TN F-a.
`See also W089/06692 published July 27, 1989. The anti-Erb82 antibodies discussed in Hudziak et a/. 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. lmmunol. 11 (3):117-127 (1991 ); Kumar et al. Mol Cell. Bioi.
`11(2):979-986 (1991); Lewis et al. Cancer lmmunol. lmmunother. 37:255-263 (1993); Pietras et al. Oncogene 9:
`1829-1838 (1994); Vitetta et al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Bioi. Chem. 269(20):
`14661-14665 (1994); Scott et al. J. Bioi. Chem. 266:14300-5 (1991); and D'souza et al. Proc. Natl. Acad. Sci. 91:
`7202-7206 (1994).
`[0006] Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe two antibodies which were 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 Erb82, and inhibit tumor cell growth in vitro.
`[0007] McKenzie et al. Oncogene 4:543-548 (1989) generated a panel of anti-ErbB2 antibodies with varying epitope
`specificities, including the antibody designated TA 1. This TA 1 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 TA 1 antibody induced maturation of the breast cancer cell lines AU-565 (which overexpresses the
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`erb82 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.
`[0008] Stancovski et al. 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 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 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 induce cell lysis in vitro via complement-dependent cytotoxicity (CDC) and antibody-mediated cell-depend(cid:173)
`ent 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.
`[0009] Bacus et al. Cancer Research 52:2580-2589 (1992) further characterized the antibodies described in Bacus
`eta/. (1990) and Stancovski eta/. of the preceding paragraphs. Extending the i.p. studies of Stancovski eta/., 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. Bacus eta/. 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 eta/. 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 N 12, N29 and N24 antibodies correlated with the extent of
`differentiation they induced.
`[001 0] Xu et al. Int. J. Cancer 53:401-408 (1993) evaluated 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 combinations), modulate cell-surface ErbB2, and inhibit ligand stimulated anchorage(cid:173)
`independent growth. See also W094/00136 published Jan 6, 1994 and Kasprzyk et at Cancer Research 52:2771-2776
`(1992) concerning anti-ErbB2 antibody combinations. Other anti-ErbB2 antibodies are discussed 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. Bioi. Chem. 267:15160-15167 (1992).
`[0011] A recombinant humanized anti-ErbB2 monoclonal antibody (a humanized version of the murine anti-ErbB2
`antibody 4D5, referred to as rhuMAb HER2 or HERCEPTIN®) has been clinically active in patients with ErbB2-overex(cid:173)
`pressing metastatic breast cancers that had received extensive prior anti-cancer therapy (Baselga et al., J. Clin. On col.
`14:737-744 [1996]).
`[0012] ErbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with
`primary disease that involves axillary lymph nodes (Siamon eta/., [1987] and [1989], supra; Ravdin and Chamness,
`Gene 159:19-27 [1995]; and Hynes and Stern, Biochim Biophys Acta 1198:165-184 [1994]), and has been linked to
`sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamde,
`methotrexate, and fluoruracil) and anthracyclines (Baselga et al., Oncology 11 (3 Suppl 1 ):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 (/big). rhuMab HER2
`was shown to enhance the activity of paclitaxel (T AXOL ®) 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 of ASCO, Vol. 13, Abstract 53 [1994]).
`
`Summary of the Invention
`
`[0013] The present invention concerns the treatment of disorders characterized by overexpression of ErbB2, 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(cid:173)
`effect of anthracycline derivatives is increased by the administration of anti-ErbB2 antibodies.
`[0014] Accordingly, the invention provides the subject-matter of the claims.
`[0015] The chemotherapeutic agent is a taxoid, such as T AXOL ® (paclitaxel) or aT AXOL ®derivative.
`[0016] Although an anti proliferative effect is sufficient, in a preferred embodiment, the anti-ErbB2 antibody is capable
`of inducing cell death or is capable of inducing apoptosis. More preferably, the antibody is the antibody 4D5, most
`preferably in a humanized form.
`[0017] The present invention is composition or manufacture of a medicament for the treatment of breast cancer,
`characterized by the overexpression of the ErbB2 receptor as claimed.
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`[0018] The medicament may be provided within 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 characterized by overexpression of ErbB2 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
`
`[0019]
`
`Fig. 1 shows epitope-mapping of the extracellular domain of ErbB2 as determined by truncation mutant analysis
`and site-directed mutagenesis (Nakamura et al. J. of Virology 67(1 0):6179-6191 [Oct 1993]; Renz et al. J. Cell Bioi.
`125(6): 1395-1406 [Jun 1994]). The anti-proliferative MAbs4D5 and 3H4 bind adjacent to the transmembrane domain.
`The various 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 SV40 termination and polyadenylation signals
`located downstream of the inserted eDNA. Plasmid DNA was transfected into 293S cells. One day following trans(cid:173)
`fection, the cells were metabolically labeled overnight in methionine and cysteine-free, low glucose DMEM containing
`I %dialyzed fetal bovine serum and 25 f.LCi 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 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 of ErbB2 (SEQ ID N0:1). 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).
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`I. Definitions
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`[0020] The
`the
`indicated otherwise,
`interchangeably. Unless
`terms "HER2", ErbB2" "c-Erb-B2" are used
`terms "ErbB2" "c-Erb-B2" and "HER2" when used herein refer to the human protein and "her2", "erbB2" and "c-erb-82"
`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).
`[0021] The "epitope 4D5" is the region in the extracellular domain of ErbB2 to which the antibody 4D5 (ATCC CRL
`1 0463) 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 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).
`[0022] 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.
`[0023] The "epitope 7C2/7F3" is the region at the N 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 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 of ErbB2; SEQ ID N0:2).
`[0024] The term "induces cell death" or "capable of inducing 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 "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 cell. In vitro, the cell may be a
`SKBR3, BT47 4, 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
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`(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),
`try pan 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".
`[0025] 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 endo(cid:173)
`plasmic 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, 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 as(cid:173)
`sociated 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 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 of annexin binding relative to untreated cell in an "annexin binding assay using
`BT474 cells" (see below).
`"Heregulin" (HRG) when used herein refers to a polypeptide which activates the ErbB2-ErbB3 and ErbB2-
`[0026]
`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. The term includes biologically active fragments and/or variants of a naturally occurring HRG polypeptide, such
`as an EGF-Iike domain fragment thereof (e.g. HRG01 177_244).
`[0027] The "ErbB2-ErbB3 protein complex" and "ErbB2-ErbB4 protein complex" are non covalently associated oligom-
`ers 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. Bioi. Chem., 269(20):14661-14665 (1994).
`"Antibodies" (Abs) and "immunoglobulins" (lgs) are glycoproteins having the same structural characteristics.
`[0028]
`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" are usually heterotetrameric glycoproteins of about 150,000
`[0029]
`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.
`[0030] 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 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 called the framework region (FR). The
`variable domains of native heavy and light chains each comprise four FR regions, largely adopting a 0-sheet configuration,
`connected by three CDRs, which form loops connecting, and in some cases forming part of, the 0-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 etal., NIH Publ. 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.
`[0031] Papain digestion of antibodies produces two identical antigen-binding fragments, called "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'h 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
`[0032]
`region consists of a dimer of one heavy- and one light-chain variable domain in 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
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`of the VwVL 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.
`[0033] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1) 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 CH 1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation
`herein for Fab' in which the cysteine residue(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.
`[0034] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two
`clearly distinct types, called kappa (K) and lambda(/<.), based on the amino acid sequences of their constant domains.
`[0035] 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: lgA, lgD, lgE, lgG, and lgM, and
`several of these may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA, and lgA2. The
`heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, o, s, y, and fL,
`respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are
`well known.
`[0036] The term "antibody" is used in the broadest sense and specifically covers intact monoclonal antibodies, poly(cid:173)
`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 intact antibody, preferably the antigen binding or variable region
`[0037]
`of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'h, and Fv fragments; diabodies; linear
`antibodies (Zapata et al. Protein Eng. 8(1 0): 1057-1062 [1995]); single-chain antibody molecules: and multispecific an(cid:173)
`tibodies formed from antibody fragments.
`[0038] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially
`homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible
`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 preparations 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 immunoglobulins. The
`modifier "monoclonal" indicates the character 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 eta I., Nature, 256:495 (1975), or may be made by recombinant DNA methods
`(see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries
`using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Bioi., 222:581-597
`(1991), for example.
`[0039] 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
`(U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).
`"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin
`[0040]
`chains or fragments thereof (such as Fv, Fab, Fab', F(ab'h or other antigen-binding subsequences of antibodies) which
`contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human
`immunoglobulins (recipient antibody) in which residues 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 immu(cid:173)
`noglobulin 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 CDRs
`correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human
`immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin
`constant region (Fe), 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. Op. Struct. Bioi., 2:593-596